Inhibitor composition for pancreatic ductal adenocarcinoma

Abstract

An inhibitor composition for pancreatic ductal adenocarcinoma, comprising an AhR inhibitor and a PRMT5 inhibitor. The AhR inhibitor can be CH223191, and the PRMT5 inhibitor can be GSK3326595. By first using a combination of small molecule inhibitors - an AhR inhibitor and a PRMT5 inhibitor, the present invention significantly inhibits the progression of pancreatic tumors in mice.

Description

Technical Field

[0001] The present invention generally relates to a small molecule inhibitor composition for pancreatic ductal adenocarcinoma. Background Art

[0002] Pancreatic ductal adenocarcinoma (PDAC) is highly malignant with extremely poor prognosis, and the average five-year survival rate is less than 10%. Its pathogenesis remains unclear. Surgery and adjuvant chemotherapy can help some patients prolong their survival, but it is only applicable to a small number of patients with resectable tumors. Existing chemotherapy such as the FORFIRINOX regimen or the gemcitabine combined with albumin-bound paclitaxel regimen has very limited improvement in the survival of most patients with unresectable tumors and is significantly associated with toxic and side reactions. Except for less than 1% of patients with microsatellite instability, most PDAC patients are difficult to benefit from existing immunotherapy regimens. The reason is related to the histological characteristics of PDAC: typical PDAC shows an immune "cold tumor" - that is, it presents an inhibitory immune microenvironment, lacking CD8 + T cells with killing function and other helper cells for adaptive immune responses. There are also obstacles in the recruitment of natural immune cells such as antigen-presenting cells, macrophages, and natural killer cells. Therefore, PDAC shows the characteristics of immunotherapy resistance, and tumors cannot be effectively recognized and killed by adaptive immune responses.

[0003] Improving the immune microenvironment of PDAC helps to improve the degree of immunotherapy response, thereby effectively inhibiting tumor progression. Currently, the academic community mainly focuses on ways such as enhancing the function of endogenous T cells, initiating anti-tumor specific T cell immune responses, and improving stromal components to facilitate the recruitment of immune cells to achieve the regulation of the tumor microenvironment (TME). Tumor cells are the source of anti-tumor immune responses. Identifying tumor endogenous antigens is crucial for initiating local immune responses and recruiting tumor-specific immune cells, and this process is one of the key ways to enhance the efficacy of immune checkpoint inhibitors.

[0004] Immune surveillance not only depends on the expression of tumor antigens by tumor cells, but more importantly on the effective presentation of tumor antigens by tumor cells to immune cells through the major histocompatibility complex (MHC). The lack of expression or dysfunction of MHC molecules is one of the important mechanisms for tumors to escape immune surveillance. The main causes of MHC molecule abnormalities include loss of heterozygosity and somatic mutations. Abnormal histone function has also been proven to be an important factor leading to the dysfunction of MHC class I tumor cell antigen presentation. For example, abnormal trimethylation of histone H3K27 inhibits the expression of MHC class I molecules in leukemia and head and neck squamous cell carcinoma. Abnormal methylation of the promoter or enhancer regions of human leukocyte antigen (HLA) genes, B2M genes, and Tap1 genes can lead to abnormal transcription of MHC-related genes, thereby weakening the tumor antigen presentation function related to MHC class I molecules. The dysregulation of promoter methylation of genes such as the type II transactivator protein gene and CD74 can lead to the loss of MHC class II molecule expression in tumor cells.

[0005] Aryl hydrocarbon receptor (AhR) is an important nuclear transcription factor that participates in processes such as cell proliferation and differentiation, stress and metabolism, and inflammatory responses, regulating the expression of multiple genes. It has now been proven to be closely related to the occurrence and development of various tumors. As a type of ligand-activated nuclear transcription factor, in the classical activated AhR signaling pathway, the AhR protein can bind to tryptophan metabolites, arachidonic acid metabolites, indole and other substances, recognize specific gene binding sequences, and thus initiate the downstream signal transduction process. AhR is very closely related to the functions of immune cells. On the one hand, it can affect the functions of innate immune cells such as macrophages and dendritic cells. On the other hand, it can also affect immune regulation by influencing the differentiation of T helper cells such as Th17 and Th22 cells, and can directly change the efficacy of CD4 + and CD8 + T cells in the adaptive immune response. Previous research results have found that there are locally high levels of AhR ligands in the tumor microenvironment: the tryptophan metabolite kynurenine (KYN). After the AhR in dendritic cells and regulatory T cells is activated, it induces immune tolerance, the content of immunosuppressive cytokines such as interleukin-10 in the immune microenvironment increases, and the function of T cells is also inhibited due to the activation of the AhR signal.

[0006] It has been found that the use of indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors can inhibit the AhR activity in PDAC tissues and cells and improve the immunosuppressive state of PDAC mouse models. There are also reports of high AhR activity in PDAC tissues. Knockout of AhR in bone marrow cells leads to an increase in the number and enhanced function of dendritic cells around the tumor, CD8 + INF-γ +The above studies reveal that AhR has an immunosuppressive effect on PDAC. However, its mechanism research is still mainly based on the immune cell components in the TME, and it is unable to explain the effect of high expression of AhR in tumor cells themselves on the formation of immunosuppressive TME, and the role of AhR in tumors in immune escape is still unclear, especially for tumor-killing specific CD8 + The mechanism of action of T cells needs to be further elucidated and utilized. Summary of the invention

[0007] The purpose of the present invention is to provide a pharmaceutical composition capable of effectively inhibiting pancreatic ductal adenocarcinoma.

[0008] According to a first aspect of the present invention, a pharmaceutical composition is provided, comprising an aryl hydrocarbon receptor (AhR) inhibitor and an arginine methyltransferase 5 (PRMT5) inhibitor.

[0009] According to the present invention, the AhR inhibitor may be CH223191, and the PRMT5 inhibitor may be GSK3326595.

[0010] The inhibitor of the present invention can be in any suitable dosage form. For example, CH223191 can be in an injection form, and GSK3326595 can be in an oral form.

[0011] According to another aspect of the present invention, provided is the use of an AhR inhibitor and a PRMT5 inhibitor in preparing a medicament for treating pancreatic ductal adenocarcinoma.

[0012] Through cell experiments and animal models based on AhR knockout tumor cells, this application found that AhR does not affect the proliferation of tumor cells themselves, but reduces the immunogenicity of PDAC by affecting tumor antigen presentation, thereby reducing the infiltration of immune cells in the tumor microenvironment and escaping the specific killing of immune cells. Specifically, AhR inhibits tumor antigen presentation of MHC class I molecules and regulates the expression of key proteins, which ultimately leads to the immune escape of PDAC. Molecular mechanism studies have found that AhR inhibits the chromatin opening of MHC class I molecules and key molecular genes by interacting with the arginine methyltransferase PRMT5, thereby inhibiting gene expression.

[0013] Therefore, this application discovered the AhR-PRMT5-MHCⅠ molecular expression pathway for the first time, thereby revealing a new molecular mechanism of pancreatic cancer tumor immune escape: AhR interacts with PRMT5 to inhibit chromatin opening, thereby inhibiting the expression of MHCⅠ molecules in tumor cells.

[0014] The present application further discovered that combined targeting of AhR and PRMT5 proteins can significantly inhibit the growth of pancreatic tumors in mice.

[0015] Due to its "cold tumor" characteristics, pancreatic cancer is insensitive to the treatment of existing immune checkpoint inhibitors (such as PD-1 antibodies). In the present invention, by first using a combination of small molecule inhibitors (AhR inhibitor and PRMT5 inhibitor), the progression of pancreatic tumors in mice was significantly inhibited. Description of the Drawings

[0016] Figure 1A It is a comparison chart of AhR expression levels in human PDAC tissues and normal pancreatic tissues in the TCGA and GTEx databases; Figure 1B It is a comparison chart of AhR expression levels in human PDAC tissues and normal pancreatic tissues in the GEO database; Figure 1C It is a survival analysis chart of PDAC patients in the TGCA database based on AhR expression levels.

[0017] Figure 2A It is an immunohistochemical staining chart of AhR in adjacent normal pancreatic tissues; Figure 2B It is an immunohistochemical staining chart of AhR in PDAC tumor tissues; Figure 2C It is an immunohistochemical staining score chart.

[0018] Figure 3A and Figure 3B respectively are Ahr - / - and wild-type Pan02 were subcutaneously inoculated into immunocompetent mice, and the tumor pictures and mouse survival time curve graphs formed by wild-type and Ahr gene tumor cells; Figure 3C 、 Figure 3D and Figure 3E respectively are Ahr - / - and wild-type PANO2 cells were orthotopically inoculated into the pancreatic tissues of immunocompetent mice. Before and after knocking out Ahr gene, the tumor formation pictures, H&E staining pictures of tumors formed by wild-type cells, Ahr H&E staining pictures of pancreatic tissues after inoculation with knocked-out cells; Figure 3F and Figure 3G respectively are Ahr - / - and the tumor pictures and nude mouse survival time curve graphs formed after subcutaneous inoculation of wild-type Pan02 cells into nude mice.

[0019] Figures 4A - 4D They are respectively histochemical staining charts of different immune cells in subcutaneous inoculated mouse tumor tissues; Figure 4E and Figure 4F They are respectively immunocyte staining count statistical charts; Figure 4G and Figure 4H They are respectively the flow cytometry detection results of activated CD4 and CD8 T cells in mouse tumor tissues; Figure 4I andFigure 4J It is Figure 4G and Figure 4H Statistical chart of flow cytometry detection results

[0020] Figure 5A and Figure 5B respectively show the expression levels of the cell membrane MHC I subunit B2M and H2-Kb; Figure 5C and Figure 5D respectively show the relative fluorescence intensities of the expressions of B2M and H2-Kb.

[0021] Figure 6A Shows the protein after IP; Figure 6B Is the typical peptide peak map of PRMT5 obtained by mass spectrometry detection; Figure 6C Shows the co-expression of AhR and PRMT5 in 293T, and CoIP detects the interaction between the two proteins; Figure 6D Is for interference Prmt5 After RNA, QPCR detects the mRNA expressions of MHC I and its key regulatory molecules; Figure 6E and Figure 6F Are respectively the comparison charts of PRMT5 expression levels in human PDAC tissues and normal pancreatic tissues in the TCGA, GTEx, and GEO databases; Figure 6G Is the survival analysis chart of PDAC patients in the TGCA database based on PRMT5 expression levels; Figure 6H and Figure 6I Are respectively the PRMT5 immunohistochemical staining charts of adjacent normal pancreatic tissues and PDAC tissues; Figure 6J Is the immunohistochemical staining score chart.

[0022] Figure 7A Shows pictures of subcutaneous tumors of mouse PDAC treated with two inhibitors alone and in combination, Figure 7B Is the corresponding statistical result of tumor weight; Figure 7C Shows the survival analysis of subcutaneous tumors of mouse PDAC treated with two inhibitors alone and in combination; Figure 7D , Figure 7E , Figure 7F and Figure 7G respectively show CD4 + T lymphocytes in the mouse PDAC subcutaneous inoculated tumor tissues of the control group, the two inhibitor treatment groups alone and in combination detected by ISH; Figure 7H Is for Figures 7D - 7E CD4 in + T lymphocyte count statistical result; Figure 7I , Figure 7J , Figure 7K and Figure 7LSeparate immunohistochemistry (ISH) assays were performed to detect CD8 + T lymphocytes in tumor tissues subcutaneously inoculated with PDAC in mice of the control group, the two inhibitor single-treatment groups, and the combination-treatment group; Figure 7M The Figures 7I - 7L CD8 + T lymphocyte count statistical results are shown in Detailed implementation manners

[0023] The present invention will be described in detail below with reference to the accompanying drawings. Those skilled in the art should understand that the embodiments described below are only exemplary descriptions of the present invention and are not used to make any limitations thereto.

[0024] Experimental procedures and analysis

[0025] 1. High expression of AhR in pancreatic cancer tumor cells regulates tumor growth through the immune system

[0026] Figure 1A is a comparison chart of AhR expression levels in human PDAC tissues and normal pancreatic tissues in the TCGA and GTEx databases; Figure 1B is a comparison chart of AhR expression levels in human PDAC tissues and normal pancreatic tissues in the GEO database; Figure 1C is a survival analysis chart of PDAC patients in the TGCA database based on AhR expression levels. From Figure 1A and Figure 1B , it can be seen that in PDAC tumor tissues compared with normal pancreatic tissues, Ahr the mRNA expression level is significantly increased. From Figure 1C , it can be seen that among PDAC patients, those with high expression of Ahr mRNA have a worse survival period.

[0027] Further, paraffin sections of 34 PDAC patients were detected: Figure 2A is an immunohistochemical staining chart of AhR in adjacent normal pancreatic tissues; Figure 2B is an immunohistochemical staining chart of AhR in tumor tissues of PDAC patients; Figure 2C is an immunohistochemical staining score chart. It can be seen that immunohistochemistry shows that AhR expression is significantly increased in tumor cells in PDAC tissues, and positive staining is shown in the tumor cell nuclei. The immunohistochemical score of the expression in tumors is higher than that of normal pancreatic ducts in adjacent tissues. The above results reveal that AhR is highly expressed in tumor cells in human PDAC tissues, and patients with its high expression have a poor prognosis. Therefore, it is speculated that highly expressed AhR in pancreatic ductal cell carcinoma plays a specific function in the process of tumorigenesis and development.

[0028] To verify the role of AhR in PDAC,Ahr Gene

[0029] Subcutaneous inoculation of C57 / BL6 mice Ahr + / + and Ahr - / - Pan02 cells, 1x10 6 cells / mouse; Figure 3A Shows the corresponding tumor pictures after knocking out the Ahr gene, Figure 3B then shows the corresponding survival curve of the mice. Figure 3C Then shows the in-situ inoculation of C57 / BL6 mouse pancreatic tissue Ahr + / + and Ahr - / - Pan02 cells, 1x10 6 cells / mouse to form the corresponding tumor pictures, Figure 3D and Figure 3E respectively show the H&E staining pictures of wild-type tumors inoculated in situ in C57 / BL6 mice, Ahr H&E staining pictures of knockout tumors. Subcutaneous inoculation of nude mice Ahr + / + and Ahr - / - Pan02 cells, 1x10 6 cells / mouse, Figure 3F shows the corresponding subcutaneous tumor pictures. Figure 3G Shows the subcutaneous inoculation of nude mice Ahr + / + and Ahr - / - The corresponding survival curve of Pan02 mice.

[0030] Inject Ahr - / - and wild-type Pan02 subcutaneously into immunocompetent C57 / BL6 mice. After knocking out the Ahr gene, the tumor volume decreases, see Figure 3A ; In addition, the survival time of the AhR gene knockout group mice is significantly prolonged, see Figure 3B . To better simulate the tumor microenvironment in situ in the pancreas and the in vivo anti-tumor immune process, it was found in the established mouse PDAC in-situ tumor model that Ahr the tumorigenicity of the knocked-out cells is significantly inhibited, and only 1 / 5 of the mice showed visible tumor masses, and no tumor masses were found in the remaining mice, see Figure 3C ; H&E staining showed that the tumors formed by wild-type cells had unclear boundaries with the pancreatic tissue, and obvious acinar duct metaplasia in the pancreatic tissue, see Figure 3D; The tumors formed by AhR knockout cells had an obvious boundary with the pancreatic tissue, and were surrounded by a large number of infiltrating stromal cells. No obvious acinar ductal metaplasia was observed. See Figure 3E . AhR knockout did not affect the growth of subcutaneous tumors in immunodeficient mice, that is, knocking out AhR had no obvious effect on the appearance of tumors, the growth rate of tumor volume, and tumor weight. Moreover, knocking out AhR could not extend the survival time of tumor-bearing nude mice. See Figure 3F and Figure 3G .

[0031] 2. Knocking out Ahr in tumor cells increased the infiltration of immune cells in the tumor microenvironment

[0032] Figure 4A shows the IHC staining of CD8 Ahr + / + T lymphocytes in subcutaneous tumors of C57 / BL6 mice after subcutaneous inoculation with Pan02 tumor cells. + Figure 4B shows the IHC staining of CD8 Ahr - / - T lymphocytes in subcutaneous tumors of C57 / BL6 mice after subcutaneous inoculation with Pan02 tumor cells. + Figure 4C shows the IHC staining of CD4 Ahr + / + T lymphocytes in subcutaneous tumors of C57 / BL6 mice after subcutaneous inoculation with Pan02 tumor cells. + Figure 4D shows the IHC staining of CD4 Ahr - / - T lymphocytes in subcutaneous tumors of C57 / BL6 mice after subcutaneous inoculation with Pan02 tumor cells. + Figure 4E shows the statistical count of CD8 Ahr + / + and Ahr - / - T lymphocytes after IHC staining of tumor tissues in C57 / BL6 mice subcutaneously inoculated with Pan02 cells. + Figure 4F shows the statistical count of CD4 Ahr + / + and Ahr - / - T lymphocytes after IHC staining of tumor tissues in C57 / BL6 mice subcutaneously inoculated with Pan02 cells. + Figure 4G shows C57 / BL6 mice subcutaneously inoculated with Ahr+ / + and Ahr - / - Pan02 cells, and the ratio of CD8 + IFγ + T lymphocytes in tumor tissues was detected by flow cytometry. Figure 4H It shows the subcutaneous inoculation of Ahr + / + and Ahr - / - Pan02 cells, and the ratio of CD4 + IFγ + T lymphocytes in tumor tissues was detected by flow cytometry. Figure 4I It shows Figure 4G the statistical results of flow cytometry detection; Figure 4J It shows Figure 4H the statistical results of flow cytometry detection.

[0033] To clarify the immunological mechanism by which knocking out tumor cells Ahr inhibits tumor growth, the above immunohistochemical staining of immune cells was performed on mouse tumor tissues. It can be seen that Ahr - / - in the tumors formed by subcutaneous inoculation of + CD4 + and CD8 Figures 4A - 4F T cell infiltration was significantly higher than that of wild-type cells, see Ahr - / - In the single-cell suspension of tumors, a higher proportion of CD4 + IFN-γ + and CD8 + IFN-γ + double-positive lymphocytes was detected, see Figures 4G - 4J . The above results suggest that AhR may promote PDAC by constructing an inhibitory immune microenvironment, reducing the infiltration and activation of anti-tumor immune cells.

[0034] 3. Knocking out tumor cells Ahr results in increased expression of the MHC I gene in tumor cells

[0035] The expression levels of the heavy chain of MHC class I molecules and β2-microglobulin (mainly including B2M and H2-Kb) on the cell membranes of wild-type cells and AhR-knockout cells were analyzed by flow cytometry.

[0036] Figure 5A It shows the flow cytometry analysis of wild-type and Ahr - / -Expression level of MHC I subunit B2M on the cell membrane of Pan02; Figure 5B shows the flow cytometry analysis of wild type and Ahr - / - Expression level of MHC I subunit H2-Kb on the cell membrane of Pan02; Figure 5C shows the statistical analysis of the relative fluorescence intensity of B2M expression detected by flow cytometry; Figure 5D shows the statistical analysis of the relative fluorescence intensity of H2-Kb expression detected by flow cytometry.

[0037] Figures 5A - 5D The results show that AhR knockout leads to a significant increase in the amounts of B2M and H2-Kb protein molecules on the surface of tumor cells. Therefore, it is confirmed that AhR has the effect of inhibiting the expression of MHC class I molecules on the surface of tumor cells, thus inhibiting tumor antigen presentation.

[0038] 4. AhR regulates the expression of MHC I molecules through interaction with PRMT5

[0039] Figure 6A shows the Coomassie brilliant blue staining of the protein electrophoresis gel after IP with tagged antibody, and the red box indicates the protein band corresponding to the molecular weight of AhR; Figure 6B for mass spectrometry detection Figure 6A the protein after IP in Figure 6C shows the co-expression of AhR and PRMT5 in 293T, and CoIP is used to detect the interaction between the two proteins. Figure 6D shows the QPCR detection of the mRNA expression of MHC I and key regulatory molecules after interfering with the expression of Prmt5 gene. Figure 6E and Figure 6F are respectively the comparison charts of PRMT5 expression levels in human PDAC tissues and normal pancreatic tissues in TCGA, GTEx, and GEO databases; Figure 6G is the survival analysis chart of PDAC patients based on PRMT5 expression level in the TGCA database. Figure 6H and Figure 6I are respectively the immunohistochemical staining charts of PRMT5 in the normal pancreatic tissues adjacent to PDAC and tumor tissues; Figure 6J is the immunohistochemical staining score chart.

[0040] To explore the molecular mechanism of AhR regulating MHC class I molecule expression, an AhR recombinant plasmid containing two protein purification and detection tags (Streptavidin and FlagM2) was constructed. After transfection of this plasmid into 293T cells, stable transfected mixed cells were screened with puromycin. After amplification, total proteins were collected for tandem affinity purification of tagged AhR. After SDS-PAGE electrophoresis and Coomassie blue staining of the purified protein, a distinct blue-stained and concentrated protein band was obtained in the region corresponding to the molecular weight of AhR, demonstrating good protein purification results. See Figure 6A . Subsequently, the gel was cut and subjected to protein mass spectrometry analysis. Analyzing the mass spectrometry results, it was found that the protein with the highest abundance related to chromatin activity was Protein arginine N-methyltransferase 5 (PRMT5), and a total of 9 types of peptide segments were captured, with a Sum PEP Score of 31.165. See Figure 6B . To verify the interaction between AhR and PRMT5, IP was performed on 293T stable transfected cells. The results showed that AhR could pull down PRMT5, demonstrating their binding and interaction. See Figure 6C .

[0041] To confirm that AhR is achieved through the interaction with PRMT5, a pancreatic ductal adenocarcinoma (PDAC) cell line was treated with its small interfering RNA (siRNA). The results showed that after PRMT5 was inhibited, the mRNA expression levels of MHC class I molecules and their key regulatory molecules were significantly increased as detected by qPCR. See Figure 6D . This indicates that AhR regulates the expression of MHC class I molecules through PRMT5.

[0042] Analysis of data in public databases TCGA, GTEx, and GEO found that in PDAC tumor tissues compared with normal pancreatic tissues, Prmt5 the mRNA expression level was significantly increased. See Figure 6E and Figure 6F ; in PDAC patients, those with high expression of Prmt5 mRNA had a worse survival. See Figure 6G .

[0043] Further examination of paraffin sections from 34 PDAC patients showed that immunohistochemistry revealed a significant increase in PRMT5 expression in tumor cells of PDAC tissues, and it was mostly positively stained in the tumor cell nuclei. The immunohistochemical score was higher than that of normal pancreatic ducts in adjacent tissues. See Figures 6H - 6JThe above results revealed that PRMT5 was highly expressed in tumor cells of human PDAC tissues, and patients with its high expression had poor prognosis. Therefore, it was speculated that highly expressed AhR and PRMT5 in pancreatic ductal carcinoma played specific functions in the process of tumorigenesis and development.

[0044] 4. Combining an AhR inhibitor and a PRMT5 inhibitor can significantly inhibit the growth of pancreatic cancer tumors in mice

[0045] The AhR inhibitor CH223191 (MCE, USA, #HY-12684) was used. The dissolution method was to sequentially add 10% DMSO + 40% PEG300 + 5% Tween80 + 45% normal saline. The working concentration was 1 mg / ml. The dosage for mice was 0.2 mg per mouse (about 20 mg / kg), and it was administered intraperitoneally three times a week for a total of 12 times. The PRMT5 inhibitor GSK3326595 (HY-101563; MedChemExpress) was administered orally at 50 mg / kg per day for a total of 15 days. Treatment started after the subcutaneous inoculated tumor grew to 0.3 cm. A total of four groups of experimental mice were set up: a blank control group (only injecting the drug solvent), an AhR inhibitor CH223191 treatment group, a PRMT5 inhibitor GSK3326595 treatment group, and a combined treatment group of the two inhibitors; there were 8 mice in each group, and two cohorts were established for each study, which were used for observing the survival curve and observing tumor tissues and biology respectively. The survival curve observation group was observed for 60 days after subcutaneous tumor formation, and the tumor tissue biology observation group was sacrificed on the 30th day after tumor formation.

[0046] Figure 7A and Figure 7B respectively show the results of the treatment of subcutaneous tumors of C57 / BL6 mice with PDAC and tumor weight statistics by the AhR inhibitor CH223191 and the PRMT5 inhibitor GSK3326595 alone and in combination. Figure 7C is the survival analysis of subcutaneous tumors of C57 / BL6 mice with PDAC treated with the AhR inhibitor CH223191, the PRMT5 inhibitor GSK3326595 alone and in combination. Figure 7D 、 Figure 7E 、 Figure 7F and Figure 7G respectively show the CD4 + T lymphocytes in the tumor tissues of C57 / BL6 mice with subcutaneous inoculated tumors of PDAC treated with the control group, the AhR inhibitor CH223191, the PRMT5 inhibitor GSK3326595 alone and in combination by ISH; Figure 7H is Figures 7D - 7E for CD4 +Statistical results of CD8 T lymphocyte counts; Figure 7I , Figure 7J , Figure 7K , Figure 7L are the CD8 T lymphocyte counts in the tumor tissues of C57 / BL6 mice with subcutaneous inoculation of PDAC tumors treated with the control group, AhR inhibitor CH223191, PRMT5 inhibitor GSK3326595 alone, and the combination of the two inhibitors by ISH detection, respectively. + T lymphocytes; Figure 7M is Figures 7I - 7L the statistical result of CD8 T lymphocyte counts in + .

[0047] In the mouse subcutaneous tumor model, after treatment with the AhR inhibitor CH223191 and the PRMT5 inhibitor GSK3326595 respectively, the tumor growth rate was significantly reduced, and after combining the two drugs GSK3326595 and CH223191, the therapeutic effect was significantly better than that of using the two drugs alone. It was manifested as smaller tumor volume, lower weight, and longer survival time of the mice. See Figures 7A - 7C . And histological examination found that there were more CD4 (see Figures 7D - 7H ) and CD8 T cell infiltration (see Figures 7I - 7M ) in the tumor tissues of the dual-drug combination treatment group. Therefore, it was confirmed in the mouse subcutaneous tumorigenesis experiment that the combination of the AhR inhibitor and the PRMT5 inhibitor could significantly inhibit the growth of pancreatic tumors inoculated in mice.

Claims

1. A pharmaceutical composition for treating pancreatic ductal adenocarcinoma, comprising an AhR inhibitor and a PRMT5 inhibitor, wherein the AhR inhibitor is CH223191 and the PRMT5 inhibitor is GSK3326595.

2. The pharmaceutical composition according to claim 1, wherein CH223191 is in an injection form and GSK3326595 is in an oral form.

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