Application of chloroquine in preparation of medicine for treating glutamine deficiency type tumor

Abstract

The invention discloses an application of chloroquine in preparation of a medicine for treating glutamine deficiency type tumors, the chloroquine promotes IFN-gamma signal transduction by up-regulating IFNGR1 protein expression, so that the therapeutic effect of immunotherapy on the glutamine deficiency type tumors, ASCT2 low expression tumors or autophagy-lysosomal pathway active tumors is enhanced. The invention provides a new thought and an effective means for solving the problem of immunotherapy drug resistance caused by glutamine metabolism limitation, and has important theoretical significance and potential clinical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to the application of chloroquine in preparing a medicine for treating glutamine-deficient tumors. Background Art

[0002] Currently, immune checkpoint inhibitors (ICIs) have been widely used in the treatment of various malignant tumors and have good application prospects. However, in actual clinical practice, only a small number of patients respond effectively to immunotherapy, and the vast majority of patients have a poor response to treatment. Studies have shown that impaired function of the gamma interferon (IFN-γ) signaling pathway in tumor cells, especially the loss or downregulation of gamma interferon receptor 1 (IFNGR1), is one of the key mechanisms leading to primary resistance to immunotherapy (Gao J, Shi LZ, Zhao H, et al. Loss of IFN-γ Pathway Genes in Tumor Cells as a Mechanism of Resistance to Anti-CTLA-4 Therapy. Cell. 2016; 167(2): 397-404.e9.).

[0003] To improve the efficacy of immune checkpoint inhibitors, existing technologies typically combine chemotherapy, radiotherapy, or targeted drugs with immune checkpoint inhibitors to enhance immune responses by enhancing antigen release and improving the tumor immune microenvironment. However, there is currently a lack of effective targeted interventions for drug resistance caused by impaired IFN-γ signaling pathways.

[0004] Previous research by the research team has found that under conditions of glutamine deficiency, tumor cells promote IFNGR1 protein degradation by inducing autophagy, inhibiting IFN-γ signaling, and ultimately leading to resistance to immunotherapy. Further studies have shown that the lysosomal inhibitor chloroquine (CQ) can significantly upregulate IFNGR1 expression in tumor cells by blocking autophagy, restoring IFN-γ signaling activity, and thus significantly increasing the sensitivity of ASCT2-low-expressing tumors to immunotherapy.

[0005] Chloroquine is an organic compound with the chemical formula C 18 H 26ClN3 is an antimalarial and anti-inflammatory agent widely used to treat malaria and rheumatoid arthritis. A clinical trial is evaluating the safety, tolerability, and efficacy of the lysosomal inhibitor hydroxychloroquine combined with the anti-PD-1 antibodies nivolumab and ipilimumab or nivolumab alone in subjects with advanced / metastatic melanoma (NCT04464759). However, there are no reports in the prior art of combining lysosomal inhibitors with immune checkpoint inhibitors for the treatment of glutamine-deficient tumors. Therefore, how to improve immunotherapy responses under the metabolic state of glutamine deficiency is a technical problem that needs to be solved urgently in this field. Summary of the Invention

[0006] The purpose of the present invention is to provide the use of chloroquine in preparing a medicine for treating glutamine-deficient tumors, provide a theoretical basis for clinical research, and be beneficial to the screening and development of therapeutic drugs.

[0007] In order to achieve the above objectives, this application adopts the following technical solutions:

[0008] In a first aspect, the present invention provides the use of chloroquine in the preparation of a medicament for treating glutamine-deficient tumors, ASCT2-low-expressing tumors, or tumors with active autophagy-lysosome pathways.

[0009] In the above technical scheme, chloroquine promotes IFN-γ signal transduction by upregulating IFNGR1 protein expression, thereby enhancing the therapeutic effect of immunotherapy on glutamine-deficient tumors, ASCT2 low-expressing tumors, or tumors with active autophagy-lysosome pathways.

[0010] In the above technical solution, chloroquine is used in combination with an immune preparation.

[0011] In the above technical solution, the drug further includes a pharmaceutically acceptable carrier, which is a filler, a wetting agent, a binder, a disintegrant or a lubricant.

[0012] In a second aspect, the present invention provides the use of chloroquine in the preparation of a drug for improving the sensitivity of glutamine-deficient tumors, ASCT2-low-expressing tumors, or autophagy-lysosome pathway-active tumors to immunotherapy.

[0013] The beneficial effects of the present invention lie in its discovery of the mechanism of action of the lysosomal inhibitor chloroquine in enhancing the response of glutamine-deficient tumors to immunotherapy. Specifically, chloroquine inhibits autophagy, preventing the degradation of IFNGR1 protein, upregulating IFN-γ signaling in tumor cells, and enhancing the response of ASCT2-low-expressing tumors to anti-PD-L1 antibody therapy. This invention provides a new approach and effective means to address the problem of immunotherapy resistance caused by limited glutamine metabolism, and has important theoretical significance and potential clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 After knocking out the glutamine transporter ASCT2 (whose encoding gene is SLC1A5) in tumor cells, the intracellular glutamine content was detected (A) and the effect of ASCT2 protein knockout on the autophagy markers LC3 and p62 was investigated by western blot (B).

[0015] Figure 2 Immunofluorescence co-localization was used to detect the co-localization relationship between IFNGR1 protein and LC3II protein in tumor cells under glutamine deficiency conditions (A), and western blot was used to investigate the effect of ASCT2 protein knockout on IFNGR1 protein expression (B).

[0016] Figure 3 Western blot was used to detect the IFN-γ signaling pathway-related protein p-STAT1 in ASCT2 protein knockout tumor cells under IFN-γ stimulation. Y701 , STAT1 and IRF1 expression levels (A) and RT-qPCR detection of the mRNA expression level of IFN-γ downstream target gene CXCL10 (B).

[0017] Figure 4 Western blot analysis of the ASCT2 protein knockout mouse lung cancer cell line LLC (A) and animal experiments to detect the therapeutic effect of anti-PD-L1 antibodies on subcutaneous tumors in ASCT2 protein knockout mice (B and C).

[0018] Figure 5 Western blot was used to detect the expression level of IFNGR1 protein in subcutaneous tumors of ASCT2 protein knockout mice (A) and IFN-γ signaling pathway-related protein p-STAT1 Y701 The expression levels of STAT1 and IFN-γ downstream target genes Cxcl10 and H2-k were detected by RT-qPCR (B) and the mRNA expression levels of IFN-γ downstream target genes Cxcl10 and H2-k were detected (C).

[0019] Figure 6Western blot analysis of the expression of IFNGR1 protein in tumor cells under the action of lysosomal inhibitor chloroquine (A) and the expression of IFN-γ signaling pathway-related protein p-STAT1 in tumor cells after combined use of IFN-γ Y701 Expression levels (B).

[0020] Figure 7 Western blot was used to detect the expression level of IFNGR1 protein in subcutaneous tumors of ASCT2 protein knockout mice under the action of chloroquine (A), and animal experiments were used to detect the therapeutic effect of chloroquine combined with anti-PD-L1 antibody on subcutaneous tumors of ASCT2 protein knockout mice (B and C). DETAILED DESCRIPTION

[0021] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the concept of the present invention to those skilled in the art. The present invention will be limited only by the claims.

[0022] The present invention discovered that knocking out the glutamine transporter ASCT2 in tumor cells can induce autophagy in tumor cells and promote the degradation of IFNGR1, thereby inhibiting IFN-γ signaling and leading to tumor cell resistance to immunotherapy. Using the lysosomal inhibitor chloroquine can inhibit autophagy, prevent the degradation of IFNGR1 protein, upregulate IFN-γ signaling, enhance the response of ASCT2-low-expressing tumors to anti-PD-L1 antibody treatment, and prolong survival.

[0023] Example 1: ASCT2 deficiency induces autophagy

[0024] siRNA was transfected using Lipofectamine 3000. Cells were transfected with si-SLC1A5 and si-NC control for 48 hours to knock down the expression of the glutamine transporter ASCT2. Intracellular glutamine levels were then measured using a glutamine assay kit. Cells were then harvested and lysed, and the expression levels of the autophagy markers LC3 and p62 were measured by Western blot to investigate the effect of ASCT2 knockdown on cellular autophagy activity.

[0025] The research results are as follows Figure 1 As shown, knockout of the tumor cell glutamine transporter ASCT2 reduced intracellular glutamine content (A); and the LC3 II protein level was significantly upregulated, while the p62 level was significantly downregulated (B), indicating that knockout of ASCT2 can induce tumor cell autophagy.

[0026] Example 2 IFNGR1 degradation depends on the autophagy-lysosome pathway

[0027] A549 cells were treated in glutamine-free medium for 12 hours, and the colocalization of LC3-II and IFNGR1 was analyzed by multiple immunofluorescence staining. Simultaneously, siRNA was transfected with Lipofectamine 3000 to knock down the expression of the tumor cell glutamine transporter ASCT2. The cells were then collected and lysed, and the expression level of IFNGR1 protein was detected by Western blot.

[0028] Immunofluorescence colocalization results Figure 2 As shown in A, under glutamine-deficient conditions, IFNGR1 and LC3-II proteins in tumor cells showed obvious co-localization signals. Western blot results are shown in Figure 2 As shown in B, knockdown of ASCT2 expression significantly downregulated IFNGR1 protein levels in tumor cells, indicating that glutamine deficiency can induce IFNGR1 protein degradation in tumor cells via the autophagy-lysosome pathway.

[0029] Example 3: ASCT2 deficiency inhibits IFN-γ signaling

[0030] Tumor cells were transfected with si-SLC1A5 and si-NC control using Lipofectamine 3000 for 48 hours, and then treated with IFN-γ (100 IU / mL) for 24 hours. The samples were collected and the cells were lysed, and p-STAT1 was detected by Western blot. Y701 , STAT1 and IRF1 protein expression levels; and after extracting tumor cell RNA, the CXCL10 gene expression level was detected by RT-qPCR.

[0031] The results are as follows Figure 3 As shown, compared with the control group, p-STAT1 Y701 , STAT1, IRF1 protein and CXCL10 gene expression were downregulated, indicating that ASCT2 protein deficiency inhibits IFN-γ signaling in tumor cells.

[0032] Example 4: ASCT2-deficient tumors are insensitive to PD-L1 inhibitors

[0033] The ASCT2 knockout LLC cell line was constructed using the CRISPR-Cas9 method, and the protein was extracted. The ASCT2 protein expression level was then detected by western blot to verify the knockout efficiency. sg-SLC1A5 LLC cells or sg-NC LLC cells (1×10 6The cells / mice were subcutaneously inoculated into the right dorsal of C57BL / 6 mice. When the tumor grew to about 75cm 3 After that, the mice were numbered and grouped, 10 mice in each group; then the mice were treated with intraperitoneal injection of drug, with or without anti-PD-L1 antibody (10 mg / kg). The tumor size was measured every two days, the longest diameter (L) and the shortest diameter (W) of the tumor were measured with vernier caliper, and the tumor volume was calculated according to the formula 0.5 x L x W 2 The tumor volume was calculated, the growth curve of tumor volume was obtained, the volume was reported as mean ± SEM, and the P value was calculated by two-way ANOVA and Tukey post-test. And the Kaplan-Meier survival curve of mice was also obtained, and the P value was determined by log-rank test.

[0034] The results are shown in Figure 4 Compared with the sg-NC group, the anti-PD-L1 antibody could not inhibit tumor growth and prolong the survival time of tumor-bearing mice in the sg-SLC1A5 group. It is shown that the treatment of PD-L1 inhibitor has no effect in the tumor with glutamine transporter ASCT2 deletion.

[0035] Example 5 ASCT2 deletion type tumor inhibits IFN-γ signaling

[0036] Further analysis of the potential mechanism of ASCT2 protein knockout mouse tumor resistance to anti-PD-L1 antibody immunotherapy. When the mouse tumor reached the endpoint, proteins and mRNAs were extracted from the subcutaneous tumor tissue of individual mice, then the tumor IFNGR1, p-STAT1 Y701 and STAT1 protein expression levels in tumor tissue were detected by western blot, and the expression of Cxcl10 and H2-k mRNA in tumor tissue was analyzed by RT-qPCR.

[0037] The results are shown in Figure 5 The expression level of IFNGR1 protein in ASCT2 protein knockout tumor is lower than that in the control group LLC tumor. And compared with the sg-NC group, the expression levels of p-STAT1 Y701 and STAT1 protein in the tumor of sg-SLC1A5 group treated with anti-PD-L1 antibody are down-regulated, and the expression of Cxcl10 and H2-k genes is also reduced. It is shown that the expression of IFNGR1 protein is down-regulated in the tumor with glutamine transporter ASCT2 deletion, and the IFN-γ signaling is inhibited.

[0038] Example 6 Chloroquine inhibits autophagy and up-regulates IFNGR1 expression

[0039] A549 cells were treated with 50 μM chloroquine (CQ) or IFN-γ (100 IU / mL) for 24 hours, and then samples were collected and cells were lysed. IFNGR1 and p-STAT1 were detected by Western blot. Y701 Protein expression levels.

[0040] Western blot results are as follows Figure 6 As shown, the lysosomal inhibitor chloroquine (CQ) upregulated IFNGR1 protein expression and promoted IFN-γ signaling.

[0041] Example 7 Chloroquine improves the sensitivity of ASCT2 low-expressing tumors to immunotherapy

[0042] sg-SLC1A5 LLC cells (1×10 6 cells / mouse) were subcutaneously inoculated into the right back of C57BL / 6 mice. 3 Afterwards, the mice were numbered and grouped, with 10 mice in each group. The mice were then treated with anti-PD-L1 antibody (10 mg / kg) combined with V9302 (40 mg / kg) or CQ (60 mg / kg) by intraperitoneal injection according to the experimental design. Tumor size and mouse weight were measured every two days. The longest diameter (L) and shortest diameter (W) of the tumor were measured with a vernier caliper. The formula 0.5×L×W was used. 2 Tumor volume was calculated, and tumor growth curves and Kaplan-Meier survival curves were obtained. When the mouse tumor growth reached the endpoint, protein was extracted from the subcutaneous tumor tissue of a single mouse, and the expression level of IFNGR1 protein in the tumor tissue was detected by western blot.

[0043] The results are as follows Figure 7 As shown, chloroquine can restore IFNGR1 expression levels in tumors with low ASCT2 expression, enhance the inhibitory effect of anti-PD-L1 antibodies on ASCT2 knockout tumors, and prolong the survival of tumor-bearing mice. This indicates that lysosomal inhibitors can improve the sensitivity of tumors with low ASCT2 protein expression to immunotherapy.

[0044] Immunotherapy has become a key treatment for non-small cell lung cancer (NSCLC), yet only a minority of patients benefit. Because solid tumors outgrow their blood supply, the tumor microenvironment is often hypoxic and nutrient-deficient. Due to the rapid consumption of tumor cells, glutamine levels in solid tumors are low.

[0045] The present invention discovered that: using lysosome inhibitors in combination with immunotherapy to treat glutamine-deficient tumors, ASCT2 low-expressing tumors or tumors with active autophagy-lysosome pathways; or applying lysosome inhibitors when combining glutamine metabolism inhibitors with immunotherapy can significantly enhance the efficacy of immunotherapy.

[0046] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. The use of chloroquine in the preparation of drugs for treating glutamine-deficient tumors, ASCT2-low-expressing tumors, or tumors with active autophagy-lysosomal pathways.

2. The application according to claim 1, characterized in that: Chloroquine promotes IFN-γ signaling by upregulating IFNGR1 protein expression, thereby enhancing the therapeutic effect of immunotherapy on glutamine-deficient tumors, tumors with low ASCT2 expression, or tumors with active autophagy-lysosome pathway.

3. The application according to claim 1, characterized in that: Chloroquine is used in combination with immune preparations.

4. The application according to claim 1, characterized in that: The drug further comprises a pharmaceutically acceptable carrier, which is a filler, a wetting agent, a binder, a disintegrant or a lubricant.

5. The use of chloroquine in the preparation of drugs for improving the sensitivity of glutamine-deficient tumors, ASCT2-low-expressing tumors, or autophagy-lysosomal pathway-active tumors to immunotherapy.

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