Use of USP1 inhibitor in the preparation of a drug for enhancing STING-mediated anti-tumor immunity

Abstract

The present invention relates to the use of USP1 inhibitors in the preparation of drugs for enhancing STING-mediated anti-tumor immunity. Through a large number of studies, the present invention has found that inhibiting the deubiquitinating enzyme USP1 can significantly enhance the response of immune cells such as DCs to dsDNA, effectively improve the activation degree of the STING-mediated type I interferon pathway, and clarify that USP1 regulates and hinders the process of STING trafficking from the endoplasmic reticulum to the Golgi apparatus through deubiquitination, thereby restricting the activation of STING. This results in insufficient efficacy of directly activating the STING pathway in tumors by means of STING direct agonists or radiotherapy and chemotherapy, and the anti-tumor effect will also be limited accordingly. The present invention reveals the mechanism of action of USP1 and the mechanism of breaking the obstructive factors, and evaluates the important role of USP1 inhibitors in enhancing STING-mediated anti-tumor immunity after radiotherapy, providing a necessary and solid foundation for the research and development of radiotherapy treatment plans and tumor immune drugs with innovative mechanisms of action, providing sufficient scientific basis for clinical translation, and having important scientific significance.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to the use of USP1 inhibitors in the preparation of drugs for enhancing STING-mediated anti-tumor immunity. Background Art

[0002] Radiation therapy (RT) for tumors is a treatment method that uses high-energy rays to cause DNA damage to tumor cells and kill them. It is an effective treatment method for controlling and ablating solid tumors in current clinical practice. According to incomplete statistics, about 70% of patients with malignant tumors need to receive radiotherapy at different stages of their treatment, and more than 50% of tumor patients receive radiotherapy and benefit. Therefore, radiotherapy plays an extremely important role in the treatment of malignant tumors. Especially in the treatment of nasopharyngeal carcinoma, early laryngeal cancer, cervical cancer, breast cancer and rectal cancer, radiotherapy can achieve the effect of preserving function and improving survival rate, and even cure, which cannot be achieved by other therapies. In recent years, the technological innovation of tumor radiotherapy has been changing with each passing day. In addition to the breakthrough of new technologies for ray directional delivery such as three-dimensional conformal radiotherapy (3D-CRT) and treatment plans, important progress has also been made in the research on the damage mechanism and effect law of radiotherapy.

[0003] The X (or γ) rays used in radiotherapy are ionizing radiation rays. These high-energy rays have wave-particle duality and can interact with other atoms or molecules (mainly water molecules) in cells to generate free radicals. These free radicals can diffuse a certain distance to reach and damage the key target DNA. It can be seen that DNA damage is the main biological effect of ionizing radiation. In addition, a large number of free radicals (such as reactive oxygen species) generated in cells also provide important conditions for the initiation of other programmed cell death methods.

[0004] It is worth noting that the effects of radiotherapy are not limited to the direct killing of tumor cells. Recent studies have shown that irradiated cells, as the source of cytokines or damage-associated molecular patterns (DAMPs), play a regulatory role in reshaping the tumor microenvironment or the systemic immune response, which may be a decisive factor in the effectiveness or duration of radiotherapy (recurrence). However, in clinical practice, the immune response induced by radiotherapy is generally at a low level, especially the abscopal effect is difficult to observe, indicating that the efficacy of triggering a systemic immune response by local irradiation is insufficient. The reason is that due to the intrinsic characteristics and self-regulation of tumor tissues and tumor cells, the key stages of the immune response induced by radiotherapy are inhibited in most cases. In the analysis of immunosuppressive factors after radiotherapy, combined with the research results in the existing technology, it is found that the release of genomic DNA (gDNA) or mitochondrial DNA (mtDNA) fragments of irradiated tumor cells has a low ability to activate the type I interferon pathway in dendritic (DC) cells or macrophages in the tumor microenvironment through dsDNA response, making it difficult to trigger anti-tumor immunity.

[0005] Studies have found that anti-tumor adaptive immunity does not occur spontaneously, and its initiation, maintenance, and the integration of the entire cancer-immune cycle are dominated by the innate immune response; the lack of innate immune response in the tumor microenvironment leads to abnormal functions in key links such as the recognition of tumor antigens, the processing and presentation of tumor antigens, and the communication between dendritic cells (DCs) and T cells, which is one of the important reasons for the generation of the "cold" tumor phenotype. Innate immunity is an important immune protection mechanism produced by organisms in response to the invasion of external pathogens or internal stress responses. It senses pathogen- or damage-associated molecular patterns (PAMPs or DAMPs) through pattern recognition receptors (PRRs) expressed by innate immune cells such as macrophages, thereby triggering a protective immune response of the body. In the tumor microenvironment, the innate immune response plays a crucial role in anti-tumor immune processes such as tumor detection, antigen presentation, initiation and maintenance of T cell activity, and formation of immune memory.

[0006] As the main signaling pathway for sensing cytoplasmic DNA, the innate immunity mediated by stimulator of interferon genes (STING) and the subsequent adaptive immune response are considered to be the main reasons for the generation of radiotherapy immune effects or abscopal effects. In the upstream DNA damage release and sensing link, under the action of high-energy rays, the DNA in the nucleus or mitochondria of tumor cells breaks and is released into the cytoplasm or taken up by immune cells, etc., and is captured by the cytoplasmic dsDNA sensor cyclic GMP-AMP synthase (cGAS) to synthesize the STING natural ligand cyclic GMP-AMP (cGAMP); in the middle interferon release link, cGAMP activates STING polymerization to recruit TBK1 (TANK-binding kinase 1), TBK1 phosphorylates itself and STING, then recruits interferon regulatory factor 3 (IRF3) and phosphorylates it, and the phosphorylated IRF3 dimer enters the nucleus to induce the release of type I interferon; in the terminal immune activation link, type I interferon promotes the maturation, infiltration and activation of dendritic cells (DCs), natural killer cells (NKs), CD8 + T cells, etc., trigger innate and adaptive immune responses, and promote tumor killing.

[0007] The release of type I interferon after radiotherapy mainly includes two key stages: DNA damage release and sensing, and STING activation - type I interferon release. In the early DNA damage release and sensing stage, the strong DNA damage repair ability of tumor cells reduces the generation of dsDNA caused by damage, and the lack of STING agonists leads to the blockage of type I interferon release. There have been a large number of research reports on targeting DNA damage repair mechanisms and relief. Although the research mainly focuses on the direct tumor killing effect caused by DNA damage, the various treatment methods developed by related research to block DNA damage repair will help to solve the immunosuppressive obstacle in this link.

[0008] The existence of inhibitory factors in the STING activation-interferon release pathway leads to the inability to stably trigger type I interferon release even when radiotherapy provides sufficient dsDNA. Therefore, compared to solving the problem of DNA damage repair, it is particularly important to eliminate the immunosuppression in this pathway. Currently, there are few reports on such research after radiotherapy, but some information has been revealed in the study of the STING signaling pathway. On the one hand, STING-mediated type I interferon release is a short-term response. In susceptible cells such as immune cells, after the STING pathway is activated, it will quickly trigger negative feedback regulation to inhibit the continuous occurrence of the immune response. On the other hand, upstream of STING, TREX1 has been found to reduce the detection of tumors by cGAS by degrading tumor-derived DNA, and ENPP1 can degrade cGAMP to reduce the activation level of STING. In summary, it shows that the stress-induced inhibitory factors that may exist in immune cells and affect type I interferon release are the main reasons for the lower dsDNA response. It is precisely because of the existence of these immunosuppressive factors that the dsDNA-cGAS-STING signaling pathway is disabled, the ability to release type I interferon into the tumor microenvironment is limited, and the immune response is inhibited from the source. Analyzing the internal mechanism of the disability in the STING activation-type I interferon release pathway helps to comprehensively clarify the core reasons for the generation of immunosuppression after radiotherapy, provides a scientific basis for stably triggering the abscopal effect of radiotherapy, and thus becomes a frontier key scientific problem and research difficulty in this field. Summary of the Invention

[0009] The object of the present invention is to solve the problems existing in the prior art, and thus provides the use of a USP1 inhibitor in the preparation of a drug for enhancing STING-mediated anti-tumor immunity. By using the USP1 inhibitor, it can significantly inhibit the activation of the STING signaling pathway and enhance the anti-tumor immune effect mediated by STING, providing a clear scientific basis and direction for enhancing the anti-tumor treatment effect after radiotherapy, and having important clinical significance.

[0010] To solve the above technical problems, the present invention is achieved through the following technical solutions.

[0011] The first aspect of the present invention provides the use of a USP1 inhibitor in the preparation of a drug for enhancing STING-mediated anti-tumor immunity.

[0012] Preferably, the USP1 inhibitor is selected from one or more of small molecule compounds, shRNA, sgRNA, siRNA designed based on USP1.

[0013] Preferably, the small molecule compound is selected from one or more of ML323 (CAS: 1572414-83-5), SJB2-043 (CAS: 63388-44-3), SJB3-019A (CAS: 2070015-29-9), KSQ-4279 (CAS: 2446480-97-1), C527 (CAS: 192718-06-2), and I-138 (CAS: 2098211-50-6).

[0014] The second aspect of the present invention provides the use of a USP1 inhibitor in the preparation of a drug for enhancing the immune effect after tumor radiotherapy.

[0015] Preferably, the USP1 inhibitor is selected from one or more of small molecule compounds, shRNA designed based on USP1, sgRNA, and siRNA.

[0016] Preferably, the small molecule compound is selected from one or more of ML323, SJB2-043, SJB3-019A, KSQ-4279, C527, and I-138.

[0017] Preferably, the immune effect is specifically an immune effect related to the activation of the STING signaling pathway. Radiotherapy is one of the means for activating the STING signaling pathway in tumors. Correspondingly, the USP1 inhibitor can enhance the tumor therapeutic activity mediated by the STING signaling pathway after radiotherapy.

[0018] Preferably, the STING signaling pathway is the dsDNA-cGAS-STING-TBK1-IRF3 signaling pathway.

[0019] The third aspect of the present invention provides the use of a USP1 inhibitor in the preparation of a drug for enhancing the therapeutic activity of a STING agonist.

[0020] Preferably, the USP1 inhibitor is selected from one or more of small molecule compounds, shRNA designed based on USP1, sgRNA, and siRNA.

[0021] Preferably, the small molecule compound is selected from one or more of ML323, SJB2-043, SJB3-019A, KSQ-4279, C527, and I-138.

[0022] Preferably, the STING agonist is selected from one or more of cGAMP (CAS: 1441190-66-4), diABZI (CAS: 2138299-33-7), MSA2 (CAS: 129425-81-6), S-cddA (CAS: 2447159-29-5), double-stranded DNA-ISD90, and DMXAA (CAS: 117570-53-3).

[0023] Preferably, the therapeutic activity is anti-tumor therapeutic activity.

[0024] Preferably, the anti-tumor therapeutic activity is anti-tumor therapeutic activity after radiotherapy.

[0025] The fourth aspect of the present invention provides a pharmaceutical composition for enhancing anti-tumor immunity, comprising a USP1 inhibitor and a STING agonist.

[0026] Preferably, the USP1 inhibitor is selected from one or more of small molecule compounds, shRNA designed based on USP1, sgRNA, and siRNA.

[0027] Preferably, the small molecule compound is selected from one or more of ML323, SJB2-043, SJB3-019A, KSQ-4279, C527, and I-138.

[0028] Preferably, the STING agonist is selected from one or more of cGAMP, diABZI, MSA2, S-cddA, double-stranded DNA-ISD90, and DMXAA.

[0029] Preferably, the pharmaceutical composition optionally includes a pharmaceutically acceptable carrier.

[0030] Preferably, the pharmaceutically acceptable carrier is selected from one or more of fillers, disintegrants, lubricants, binders, preservatives, antioxidants, chelating agents, colorants, flavoring agents, fragrances, and solvents.

[0031] The fifth aspect of the present invention provides the use of a USP1 inhibitor in the preparation of a product for promoting the release of type I interferon.

[0032] Preferably, the type I interferon is selected from one or more of INFα, INFβ, and INFκ.

[0033] Preferably, the USP1 inhibitor is selected from one or more of small molecule compounds, shRNA designed based on USP1, sgRNA, and siRNA.

[0034] Preferably, the small molecule compound is selected from one or more of ML323, SJB2-043, SJB3-019A, KSQ-4279, C527, and I-138.

[0035] The sixth aspect of the present invention provides the use of a USP1 inhibitor in the preparation of a drug for promoting the activation of the STING signaling pathway.

[0036] Preferably, the USP1 inhibitor is selected from one or more of small molecule compounds, shRNA designed based on USP1, sgRNA, and siRNA.

[0037] Preferably, the small molecule compound is selected from one or more of ML323, SJB2-043, SJB3-019A, KSQ-4279, C527, and I-138.

[0038] Preferably, the STING signaling pathway is the dsDNA-cGAS-STING-TBK1-IRF3 signaling pathway.

[0039] It should be understood that, unless otherwise specified, in the context of the present invention, the USP1 inhibitor refers to a substance that can specifically inhibit the activity and / or expression level of USP1. Those skilled in the art can commercially purchase or synthesize relevant small molecule USP1 inhibitors according to actual needs, or can also design and synthesize nucleic acids or proteins that can produce inhibitory activity against USP1 through molecular biology means, as long as the level and / or activity of USP1 can be reduced. The small molecule compounds such as ML323, SJB2-043, SJB3-019A, KSQ-4279, C527, and I-138 listed in the context of the present invention are all common types of USP1 inhibitors in the art, which can specifically inhibit the activity of USP1 and / or reduce its expression level. Those skilled in the art can clearly understand that the selection of the above specific USP1 inhibitors is only for demonstrating the technical solution of the present invention, so that those skilled in the art can more fully and completely understand the basic inventive concept of the present invention. The listed specific USP1 inhibitors do not limit the technical solution and protection scope of the present invention. The basic concept and main contribution of the present invention lie in clarifying the effect enhancement of USP1 inhibitors on STING-mediated anti-tumor immunity, that is, as long as the inhibition of USP1 can be achieved, the enhancement of STING-mediated anti-tumor immune effect can be achieved. Therefore, in addition to the specific USP1 inhibitors listed in the present invention, those skilled in the art can know that any other components that can inhibit the activity and / or expression level of USP1 can also achieve the enhancement of STING-mediated anti-tumor immune effect.

[0040] Existing studies have found that after injecting synthetic cGAMP-like STING agonists into transplanted tumors, the lung metastases along with the transplanted tumors disappeared, proving that activating the Sting pathway can initiate adaptive immunity through a cascade reaction to generate anti-tumor immune memory and produce abscopal effects. Nevertheless, due to the systemic distribution characteristics of immune cells, the use of STING agonists may trigger systemic immune side effects. Therefore, most current STING agonists have to be administered by intratumoral injection. As an important local treatment method, radiotherapy has obvious advantages in the process of cGAS-STING activation. It can achieve precise coverage of the tumor area while avoiding the occurrence of systemic side effects, so there is no need to use STING agonists that may cause systemic adverse reactions.

[0041] Theoretically, the DNA damage caused by radiotherapy can achieve immune activation or abscopal effects after radiotherapy through the above-mentioned dsDNA-cGAS-STING signaling pathway. However, in clinical practice, cases that can produce systemic immune effects are extremely rare because under tumor pathological conditions, the dsDNA-cGAS-STING immune pathway is dysregulated, resulting in the inhibition of type I interferon release. Therefore, to stably trigger the immune response of radiotherapy, it is necessary to specifically overcome the inhibitory factors in the upstream, middle, and downstream links of the dsDNA-cGAS-STING signaling pathway, and the analysis of related mechanisms is indispensable. However, current research on how to trigger the abscopal effect of radiotherapy mainly focuses on the terminal immune response link after the release of type I interferon. For example, the activation of immune signals after radiotherapy will induce the expression of inhibitory factors such as immune checkpoints and the cytokine TGF-β1, and various clinical trials of radiotherapy combined with immune checkpoint inhibitors, TGF-β1 antibodies, etc. have also achieved positive results. Nevertheless, the combined use of radiotherapy and immunotherapy regimens can only produce clear effects in "hot tumors" with active immune signals, but is not effective in "cold tumors" with inactive immune signals. Logically, the absence of the immune response after radiotherapy may be caused by late immune inhibitory factors such as immune checkpoints, or it may also be due to the obstruction of upstream type I interferon release, resulting in the failure to activate the immune pathway. Therefore, triggering the release of type I interferon is particularly important for achieving immune activation in tumor tissues.

[0042] USP1 (Ubiquitin carboxyl-terminal hydrolase 1) is a member of the ubiquitin-specific level enzyme family and belongs to the sulfhydryl hydrolase class. USP1 can stabilize DNA-binding inhibitory proteins (ID1 / ID2 / ID3) by deubiquitinating and regulate DNA damage repair by cleaving the monoubiquitination of FANCD2 and PCNA. In addition, USP1 can also regulate processes such as tumor progression and chemotherapy resistance by maintaining the homeostasis of ULK1, MAX, MAST1 and TAZ by deubiquitinating. However, the regulatory mechanism of USP1 on the dsDNA-cGAS-STING-IRF3 signaling pathway has not yet been revealed.

[0043] In this regard, the present invention has conducted a large number of studies, based on the basic research ideas of chemical intervention phenotype-active compound discovery-pathological / pharmacological mechanism, and carried out in-depth and systematic exploration of the key factors affecting the tumor microenvironment. More than 1,000 active molecule libraries with different structures, including deubiquitinating enzymes (DUBs), kinases (Kinases), phosphodiesterases (PDEs) and other target inhibitors, were screened in two types of schemes. One was radiotherapy-related in vitro co-incubation experiment screening, which was based on the establishment of an interactive relationship between irradiated tumor cells and dendritic cells or fibroblasts in the tumor microenvironment, and the activation of the type I interferon pathway after THP1 cells took up dsDNA secreted by tumor cells; the second was to directly use STING agonists to examine the ability of various active molecules to synergistically activate STING. The results showed that USP1 inhibitors showed excellent activity in both screening schemes, and could significantly enhance the ISG response in dendritic cells or fibroblasts. It was also verified in DC2.4 cells that knocking out USP1 could significantly enhance the responsiveness of cells to dsDNA or STING agonists, while the synergistic effect of USP1 inhibitors was lost. It can be seen that deubiquitination regulation mediated by USP1 is one of the key factors in inhibiting the type I interferon signal response in immune cells.

[0044] Generally speaking, the present invention discovers that by inhibiting the deubiquitinating enzyme USP1, the immune cells such as DCs can significantly enhance the response to dsDNA, effectively improving the activation degree of the STING-mediated type I interferon pathway. It is clarified that USP1 mediates the retrograde transport of STING from the Golgi apparatus to the endoplasmic reticulum through deubiquitination regulation, thereby hindering the activation of STING. This mechanism is completely different from the activation link of IRF3 affected by USP14. The present invention reveals in detail the mechanism of action of USP1, and evaluates the important role of USP1 inhibitors in enhancing anti-tumor immunity after radiotherapy in cell and animal models, providing a necessary and solid foundation for the development of radiotherapy treatment plans and tumor immune drugs with innovative mechanisms of action, providing sufficient scientific basis for clinical translation, and having important scientific significance. Brief Description of the Drawings

[0045] Figure 1 It is a schematic diagram of the result that the USP1 inhibitor ML323 can synergistically promote the secretion of IFN-β1 by BMDCs in the co-incubation system.

[0046] Figure 2 It is a schematic diagram of the result of the effect of the USP1 inhibitor ML323 on the secretion of IFN-β1 in DC2.4, L929, and RAW264.7 cells in the co-incubation system (H22).

[0047] Figure 3 It is a schematic diagram of the result of the effect of the USP1 inhibitor ML323 on the expression of IFN-β1 in DC2.4, L929, and RAW264.7 cells in the co-incubation system (H22).

[0048] Figure 4 It is a schematic diagram of the result of the effect of the USP1 inhibitor ML323 on IFN-β1 in DC2.4, L929, and RAW264.7 cells in the co-incubation system (CT26).

[0049] Figure 5 It is a schematic diagram of the result of the effect of ML323 on IFN-β1 and CXCL-10 in tumor-bearing mice.

[0050] Figure 6 It is a schematic diagram of the result of the effect of the USP1 inhibitor SJB2-043 on IFN-β1 in the co-incubation system (H22).

[0051] Figure 7 It is a schematic diagram of the result of the effect of SJB2-043 on IFN-β1 and CXCL-10 in tumor-bearing mice.

[0052] Figure 8 It is a schematic diagram of the result of the effect of using the USP1 inhibitor alone on tumor cells.

[0053] Figure 9 Schematic diagram showing that pre-incubation with ML323 can significantly enhance the IFN-β1 production in DC2.4 and L929 cells stimulated by LSD90.

[0054] Figure 10 Schematic diagram showing the effect of ML323 on dsDNA during co-incubation.

[0055] Figure 11 Schematic diagram showing the effect of ML323 on the cytoplasmic 2',3'-cGAMP level during co-incubation.

[0056] Figure 12 Schematic diagram showing that ML323 can exert excellent synergistic effects in DC2.4, L929, and RAW264.7 cells when the STING signaling pathway is activated.

[0057] Figure 13 Schematic diagram showing the effect of ML323 on IFN-β1 expression in DC2.4 and L929 cells under the action of S-cddA.

[0058] Figure 14 Schematic diagram showing the effect of STING inhibitor on the synergistic effect between ML323 and ISD90.

[0059] Figure 15 Schematic diagram showing the effect of STING inhibitor on the synergistic effect produced by ML323 during co-incubation.

[0060] Figure 16 Schematic diagram showing the effect of USP1 inhibitor on the synergistic effect with STING agonist.

[0061] Figure 17 Schematic diagram showing the effect of time on the synergistic effect between ML323 and S-cddA in DC2.4 cells.

[0062] Figure 18 Schematic diagram showing the effect of ML323 concentration on the synergistic effect between ML323 and S-cddA in DC2.4 cells.

[0063] Figure 19 Schematic diagram showing the effect of time on the synergistic effect between ML323 and S-cddA in L929 cells.

[0064] Figure 20 Schematic diagram showing the effect of ML323 concentration on the synergistic effect between ML323 and S-cddA in L929 cells.

[0065] Figure 21Schematic diagram of the time-dependent synergistic effect of ML323 and S-cddA in RAW264.7 cells.

[0066] Figure 22 Schematic diagram of the effect of ML323 concentration on the synergistic effect of ML323 and S-cddA in RAW264.7 cells.

[0067] Figure 23 Schematic diagram of the effect of S-cddA concentration on the synergistic effect of ML323 and S-cddA in DC2.4 cells.

[0068] Figure 24 Schematic diagram of the effect of S-cddA concentration on the synergistic effect of ML323 and S-cddA in L929 cells.

[0069] Figure 25 Schematic diagram of the effect of S-cd dA concentration on the synergistic effect of ML323 and S-cddA in RAW264.7 cells.

[0070] Figure 26 Schematic diagram of the effect of the combination of USP1 inhibitor and high-dose radiotherapy on the growth rate and volume of tumors in vivo. Detailed implementation manners

[0071] To make the objectives, technical solutions and effects of the present invention clearer and more definite, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0072] Unless otherwise specified, cell lines listed in the context of the present invention, including DC2.4 (mouse dendritic cells), RAW264.7 (mouse monocyte macrophage leukemia cells), THP 1 (human monocytic leukemia cells), H22, BMDC, etc., are cultured according to the prior art. All cell lines are identified by short tandem repeat analysis at the China Center for Type Culture Collection (Wuhan) and verified for mycoplasma contamination using a PCR detection kit (Shanghai Biothrive Sci). They are also cryopreserved in liquid nitrogen and used for subsequent experiments. Among the reagents used in the present invention, all are commercially available. The STING signaling pathway agonists are selected from SATE-cddA (S-cddA, CAS: 2447159-29-5), double-stranded DNA-ISD90, etc., and are used to stimulate and mimic the activation of the STING signaling pathway. For the convenience of demonstrating the technical solutions and concepts of the present invention, experiments related to USP1 inhibitors in the context of the present invention are all carried out using ML323, SJB2-043, SJB3-019A, KSQ-4279, C527, I-138, etc. Those skilled in the art can know that other components with inhibitory activity against USP1 have the same or similar effects and can be specifically verified using the methods mentioned in the art or in the present invention. The listing and selection of USP1 inhibitors in the present invention do not limit the present invention. The experimental methods used in the present invention, such as molecular biology experiments, cell experiments, animal experiments, etc., are all conventional methods and techniques in the art. Representative results are presented in the context drawings for biological experiment replicates, and the data are presented as mean±SD and mean±SEM as specified in the drawings. All in vitro experiments are repeated at least three times. The data are analyzed using GraphPad Prism 8.0 or SPSS22.0 software. Conventional medical statistical methods such as t-tests, chi-square tests, and analysis of variance are used to compare the mean differences between two or more groups. p < 0.05 is considered a significant difference.

[0073] Example 1

[0074] First, starting from the basic research idea of chemical intervention phenotype - active compound discovery - pathological / pharmacological mechanism, in - depth and systematic exploration was carried out on the key factors affecting the tumor microenvironment. Screening of more than 1000 active molecular libraries with different structures, including deubiquitinases (DUBs), kinases, phosphodiesterases (PDEs), and other target inhibitors, was conducted using two protocols. One was in - vitro co - incubation experiments related to radiotherapy, which was based on establishing an interaction relationship by co - incubating irradiated tumor cells with THP1 cells, and examining the activation of the type I interferon pathway after DC2.4 cells internalized dsDNA secreted by tumor cells. The other was directly using STING agonists to examine the ability of various active molecules to synergistically activate STING. The results showed that the USP1 inhibitor ML323 (CAS: 1572414 - 83 - 5) exhibited excellent activity in both screening protocols and could significantly enhance the ISG response in DC2.4 cells.

[0075] Subsequently, the USP1 inhibitor ML323 was co - incubated with H22, BMDCs, etc. to study its effect on radiotherapy - induced immune activation. Specifically, after irradiating H22 with a dose of 40 Gy, H22 was co - incubated with BMDCs, and then the supernatant was taken for Elisa detection of secreted IFN - β1, and BMDCs were subjected to qPCR for detecting IFN - β1 expression. The results showed that when the USP1 inhibitor ML323 (5 μM) was co - incubated with H22 and BMCDs cells, it could exhibit an excellent synergistic effect with radiotherapy; detection of secreted IFN - β1 produced by BMDCs using an IFN - β1 Elisa kit and qPCR detection of IFN - β1 gene expression in BMDCs indicated that the USP1 inhibitor ML323 could synergistically promote the secretion (****p < 0.0001) and expression (*****p < 0.0001) of IFN - β in the co - incubation system (see Figure 1 ).

[0076] Referring to the above experimental method, ML323 was co - incubated with H22 cells in DC2.4, L929, and RAW264.7 cells respectively, and the secretion of IFN - β1 was detected. The results showed that ML323 could promote the secretion of IFN - β1 (***p < 0.001, ****p < 0.0001) (see Figure 2 ); qPCR detection of IFN - β1 gene expression in BMDCs showed that ML323 could promote the expression of IFN - β1 in DC2.4, L929, and RAW264.7 cells (see Figure 3) Subsequently, ML323 was co-incubated with CT26 cells in DC2.4, L929, and RAW264.7 cells respectively, and the secretion of IFN-β1 was detected. The results were similar to those above, that is, ML323 could also promote the secretion of IFN-β1 (***p < 0.001, ****p < 0.0001) (see Figure 4 ).

[0077] Furthermore, CT26 was used for tumor xenotransplantation in Balb / c mice. After tumor formation, the implanted tumors were taken for radiotherapy and the secretion of IFN-β1 and CXCL-10 was detected. Specifically, CT26 cells were cultured to form tumors in the armpits of Balb / c mice (1×10^6 / mouse). After tumor formation in about one week, the complete tumors were removed, ground and enzymolyzed, and then cultured adherently; then radiotherapy and radiotherapy combined with the USP1 inhibitor ML323 (5 μM) were carried out (irradiation dose 8 Gy, continuous irradiation for three days). 24 h after radiotherapy, the supernatant was taken for Elisa to detect the secretion of IFN-β1 and CXCL 10. The results showed that ML323 could synergistically promote the secretion of IFN-β1 and CXCL-10 in mouse tumor tissues (see Figure 5 ).

[0078] In addition to ML323, another USP1 inhibitor SJB2-043 (CAS: 63388-44-3) was selected to repeat the above experiment. The results showed that SJB2-043 could also exert a certain synergistic effect when co-incubated with DC2.4, L929, RAW264.7 and H22. The IFN-β1 Elisa kit was used to detect the secreted IFN-β1 produced by BMDCs, indicating that SJB2-043 could synergistically promote the secretion of IFN-β by BMDCs in the co-incubation system (*p < 0.05, **p < 0.01) (see Figure 6 ); the in vivo experimental results showed that after radiotherapy and detection of IFN-β1 and CXCL-10 secretion after tumor formation with CT26 cells in Balb / c mice, SJB2-043 could synergistically promote the secretion of IFN-β1 and CXCL10 in mouse tumor tissues (**p < 0.05, ***p < 0.001) (see Figure 7 ). The above results together indicate that USP1 inhibitors can synergistically promote the immune activation caused by radiotherapy.

[0079] Example 2

[0080] To clarify the direct action activity of USP1 inhibitors on the radiotherapy of tumor cells, H22 and CT26 cells were pretreated with corresponding concentrations of ML323, followed by radiotherapy. After 2 - 4 hours, the cells were harvested to extract RNA, and the mRNA levels of factors such as IFN-β1 were detected by qPCR. The results showed that in the absence of immune cells, treating tumor cells with USP1 inhibitors alone was difficult to activate the STING signaling pathway and the expression and secretion of IFN-β1 in tumor cells after radiotherapy (see Figure 8 ), indicating that USP1 inhibitors are mainly used to enhance the activity of the STING pathway in immune cells and have little effect on tumor cells. After radiotherapy of tumors in vivo, tumor cells, as donors of substances such as dsDNA that activate immune cells, can effectively enhance the immunogenicity of related immune cells in tumors under the action of USP1 inhibitors.

[0081] ML323 was pre-incubated with DC2.4 and L929 cells respectively. H22 cells (10 7 / well) were taken and after irradiation treatment (irradiation dose 40 Gy), the H22 cells were resuspended with medium containing DUB inhibitors and added to BMDCs / DC2.4 / L929 / RAW 264.7 (cell number 10 7 / well) cultured adherently. After co-culturing for 12 h, the supernatant was taken for Elisa to detect the secretion of IFN-β1 and CXCL10; after washing away the H22 cells, the RNA of BMDCs was extracted and the expression of IFN-β1 and CXCL10 was detected by qPCR. The results showed that pre-incubation treatment with ML323 could significantly enhance the IFN-β1 produced by double-stranded DNA-ISD90-stimulated DC2.4 and L929 (see Figure 9 ). Detection of dsDNA and cGAMP during the co-incubation process found that ML323 did not affect the production of dsDNA and the level of 2’,3’-cGAMP in the cytoplasm during the co-incubation process (****p < 0.0001) (see Figures 10 - 11 ).

[0082] Subsequently, DC2.4, L929, and RAW264.7 cells were treated with 2’,3’-cGAMP (100 nM, 2 h) or S-cddA (100 nM, 2 h) as STING agonists respectively. After DC2.4 / L929 / RAW 264.7 cells were plated and adhered (cell number: 500,000), cGAMP (100 nM) / S-cddA (100 nM), cGAMP (100 nM) + ML323 (5 μM) / S-cddA (100 nM) + ML323 (5 μM) were added for stimulation. RNA was extracted at 2 h, and the expression of IFN-β1 and CXCL10 was detected using qPCR method. It was found that in the case of STING signaling pathway activation, ML323 could exert excellent synergistic effects in DC2.4, L929, and RAW264.7 cells (see Figure 12 ). Meanwhile, when S-cddA (100 nM, 2 h) was used as the STING agonist, ML323 could significantly stimulate the expression of IFN-β1 in DC2.4 and L929 cells in a short time (see Figure 13 ), and the EC 50 values of ML323 for promoting IFNβ1 in DC2.4 and L929 cells were 3.625 μM and 9.539 μM respectively.

[0083] In addition to 2’,3’-cGAMP and S-cddA, another STING agonist ISD90 was used to repeat the above experiment. It was found that when ISD90 was used as the agonist, after pre-incubation with ML323 in DC2.4 and L929, excellent synergistic effects could be exerted, and the EC 50 values of ML323 for promoting IFNβ1 in DC2.4 and L929 were 5.188 μM and 4.064 μM respectively.

[0084] DC2.4 and L929 cells were treated with the STING inhibitor H151 respectively. After DC2.4 / L929 were plated and adhered (cell number: 500,000), H151 (5 μM) and DMSO were used for incubation for 4 h, then ISD90 was added for stimulation. RNA was extracted at 2 h, and the expression of IFN-β1 and CXCL10 was detected using qPCR method. It was found that the synergistic effects of ML323 and ISD90 in DC2.4 and L929 disappeared (****p < 0.0001) (see Figure 14 ); meanwhile, it was also found that the synergistic effects of ML323 and co-incubation in DC2.4 and L929 cells disappeared (*p < 0.05, ****p < 0.0001) (see Figure 15 ).

[0085] Furthermore, to study the effect of USP1 inhibitor concentration on the synergy with STING agonists, after plating and adhering L929 cells (500,000 cells), one group was stimulated with ML323 + concentration gradients of DMXAA (STING agonist) (0 / 2.5 / 5 / 10 / 25 / 50 μM), and another group was stimulated with S-cddA (100 nM), S-cddA (100 nM) + concentration gradients of SJB2-043 (1 / 2.5 / 5 / 10 / 20 μM); RNA was extracted at 2 h respectively, and the expression of IFN-β1 was detected using qPCR method. The results showed that USP1 inhibitor ML323 and DMXAA, SJB2-043 and S-cddA could both exert synergistic effects, and the synergistic effects were enhanced with the increase of the concentration of DMXAA or SJB2-043, showing concentration dependence (see Figure 16 ). The above results together indicate that the synergistic effect exerted by USP1 inhibitor is cGAS-STING pathway-dependent, and this synergistic effect is concentration-dependent.

[0086] Example 3

[0087] To further study the regulatory effect of USP1 inhibitor on the cGAS-STING signaling pathway, in DC2.4 cells, using S-cddA as an agonist, the synergistic effect of ML323 and S-cddA was manifested as a change over time. After plating and adhering DC2.4 cells (1,000,000 cells), S-cddA (100 nM) and S-cddA (100 nM) + ML323 (5 μM) were added, and proteins were extracted at 0 / 2 / 4 / 6 / 8 / 10 h respectively, and the expression of P-STING and P-TBK1 proteins was detected using Weston blot method. The results showed that the levels of P-STING and P-TBK1 gradually increased (see Figure 17 ); at the same time, the synergistic effect was enhanced with the increase of the concentration of ML323, and the levels of P-STING and P-TBK1 were positively correlated with the concentration of ML323 (see Figure 18 ).

[0088] Subsequently, the above experiments were repeated in L929 and RAW264.7 cells, and similar results were also observed, that is, using S-cddA (100 nM) as an agonist, ML323 and S-cddA exerted a synergistic effect, manifested as a gradual increase in the levels of P-STING and P-TBK1 over time; at the same time, the synergistic effect was enhanced with the increase of the concentration of ML323, and the levels of P-STING and P-TBK1 were positively correlated with the concentration of ML323 (see Figures 19 - 22 ).

[0089] Furthermore, in DC2.4, L929, and RAW264.7 cells, the concentration of ML323 was fixed (5 μM), and the synergistic effect of ML323 and S-cddA was studied. The results showed that the synergistic effect was enhanced with the increase in the concentration of S-cddA, and the levels of P-STING and PTBK1 were positively correlated with the concentration of S-cddA (see Figures 23 - 25 ).

[0090] Example 4

[0091] The therapeutic activity of the USP1 inhibitor in combination with radiotherapy in vivo was studied. The specific steps were as follows: Female Balb / C mice, 4 - 5 weeks old, were inoculated with tumors in both axillae at a dose of 100 w / mouse. One week later, the mice with established tumors were separated into cages and divided into a blank control group; an ML323 group; a radiotherapy group; and a radiotherapy + ML323 group. The ML323 group and the radiotherapy + ML323 group were continuously administered ML323 (10 mg / kg) for 7 days starting from the day before radiotherapy. Starting from the 11th day, the radiotherapy group and the radiotherapy + ML323 group started radiotherapy simultaneously, and the radiotherapy dose was 8 Gy × 5 days. After radiotherapy, the tumor volume was continuously recorded. The mice in the blank control group and the ML323 group were sacrificed on the 15th day, and the mice in the radiotherapy group and the radiotherapy + ML323 group were sacrificed on the 23rd day. The tumors were removed, fixed with paraformaldehyde, and sectioned for HE and IHC staining analysis. The results showed that both radiotherapy and the radiotherapy combined with ML323 treatment groups could activate the immunity of mice under high-dose irradiation and had a control effect on tumors. Compared with the radiotherapy group, the combined ML323 treatment group could control the growth of tumors in a very short time, and after radiotherapy, the tumor control was still better than that of the radiotherapy treatment group (see Figure 26 ). It is worth noting that the control of the unirradiated distant tumors after combining with ML323 was also significantly stronger than that of the single radiotherapy group, indicating that ML323 could significantly enhance the immune effect after radiotherapy.

[0092] As can be clearly seen from the above, the deubiquitination regulation mediated by USP1 is one of the key factors inhibiting the type I interferon signaling response in immune cells. USP1 mediates the stress inhibitory factor for the production of type I interferon in susceptible cells such as immune cells, thereby hindering the failure of anti-tumor immune activation after radiotherapy. Mechanistic studies have shown that USP1 can rapidly sense the signals of STING aggregation and trafficking, remove its ubiquitinated state, and thus interrupt the continuous activation of the STING signal. Generally speaking, the present invention discovers that by inhibiting the deubiquitinating enzyme USP1, the response of immune cells such as DCs to dsDNA can be greatly enhanced, effectively improving the activation degree of the STING-mediated type I interferon pathway, and clarifying the mechanism of action of USP1. The present invention discloses in detail the mechanism of action of USP1, and evaluates the important role of USP1 inhibitors in enhancing anti-tumor immunity after radiotherapy in cell and animal models, providing a necessary and solid foundation for the development of radiotherapy treatment regimens and tumor immune drugs with innovative mechanisms of action, providing sufficient scientific basis for clinical translation, and having important scientific significance.

[0093] The above specific embodiments section specifically introduces the analysis method involved in the present invention. It should be noted that the above introduction is only to help those skilled in the art better understand the method and idea of the present invention, rather than a limitation on the relevant content. Without departing from the principle of the present invention, those skilled in the art can also make appropriate adjustments or modifications to the present invention, and the above adjustments and modifications should also fall within the protection scope of the present invention.

Claims

1. A pharmaceutical composition for enhancing anti-tumor immunity, characterized in that: Including USP1 inhibitors and STING agonists; The USP1 inhibitor is selected from small molecule compounds, and the small molecule compound is selected from one or more of ML323 and SJB2-043; The STING agonist is selected from one or more of 2', 3'-cGAMP, S-cddA, double-stranded DNA-ISD90, and DMXAA; wherein the CAS number of S-cddA is 2447159-29-5.

2. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition includes a pharmaceutically acceptable carrier.

3. The pharmaceutical composition according to claim 2, characterized in that The pharmaceutically acceptable carrier is selected from one or more of a filler, a disintegrant, a lubricant, a binder, a preservative, an antioxidant, a chelating agent, a colorant, a flavoring agent, an aromatic agent, and a solvent.

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