Application of MED12 as an immunotherapy target in screening drugs for the treatment of pancreatic cancer

Abstract

The present invention provides the use of MED12 as an immunotherapy target in screening drugs for treating pancreatic cancer, and the amino acid sequence of MED12 is shown in SEQ ID NO.2. The present invention also provides the use of immunotherapy drugs or chemotherapy drugs combined with drugs targeting MED12 in the preparation of drugs for treating pancreatic cancer, and the amino acid sequence of MED12 is shown in SEQ ID NO.2. The present invention also provides the use of MED12 as a biomarker for screening immunotherapy targets in the treatment of pancreatic cancer. The present invention also provides the use of MED12 as a biomarker for screening immunotherapy targets in the treatment of pancreatic cancer in the preparation of a detection kit. Experiments have shown that With12 More tumor-killing T cells can be infiltrated into the missing tumors, and this group of T cells has stronger cytokine secretion and proliferation capabilities. With12 The absence of can effectively promote the inhibitory effect of PD-1 monoclonal antibodies on tumors and significantly prolong the survival of tumor-bearing mice.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to immunotherapy screening targets, specifically to the application of MED12 as an immunotherapy screening target in the treatment of pancreatic cancer. Background Art

[0002] Immunotherapy is a cancer treatment that enhances the patient's own immune system to eliminate cancer cells. It is a treatment method that actively or passively causes the body to produce tumor-specific responses and exerts its function of inhibiting and killing tumor cells. Immunotherapy mainly includes tumor vaccine therapy, oncolytic virus therapy, dendritic cell immunotherapy (DC therapy), adoptive cell therapy (ACT), antibody-drug conjugates (ADC) and immune checkpoint inhibitors (ICIs). Among them, ICIs using PD-1 and PD-L1 are the most widely used and successful immunotherapy methods to date. Immune checkpoint inhibitor therapy has very significant efficacy in many malignant solid tumors, can effectively inhibit tumor growth and prolong patient survival. The mechanism of action of immune checkpoint inhibitors is mainly to restore the immune activity of patients' T cells by counteracting immunosuppressive receptors such as CTLA-4, PD-1 or PD-L1, thereby enhancing anti-tumor immune response. Current studies have found that people who respond well to PD-1 antibodies and PD-L1 antibodies mainly include those with high tumor gene mutation load TMB, a large number of immune cells infiltrating the tumor tissue, microsatellite instability (MSH-I), and patients with smaller tumor size, younger age, and better physical condition.

[0003] Pancreatic cancer, the "king of cancers", presents typical "cold tumor" characteristics due to its low tumor mutation load, poor immunogenicity, high immunosuppression and high fibrosis of the tumor microenvironment. At present, there are still great challenges in the application of immunotherapy in pancreatic cancer. Immunotherapy represented by immune checkpoint inhibitors has almost been wiped out in pancreatic cancer, and most of them have failed in phase I and II clinical trials. At present, the NCCN guidelines for pancreatic cancer only recommend pembrolizumab as the first-line and second-line treatment for patients with advanced pancreatic cancer with MSI-H (microsatellite high instability) or dMMR (mismatch repair deficiency), but these patients account for less than 1% of pancreatic cancer. Given the effectiveness of immune checkpoint inhibitors in other cancers and the limitations of traditional treatments for pancreatic cancer, it is of great significance to explore the application of immunotherapy in pancreatic cancer.

[0004] Current studies have shown that endogenous epigenetic regulatory factors in tumors play a critical role in reshaping the immune microenvironment. Inhibiting epigenetic regulatory factors such as SETDB1, KDM5B, and PHF8 can effectively enhance the effectiveness of immune checkpoint inhibitors in melanoma and colorectal cancer.

[0005] MED12 is an important epigenetic regulatory molecule. Med12 Defects in pancreatic cancer can induce an epithelial-mesenchymal transition phenotype in tumors through the TGF-β signaling pathway, promote tumor progression, and thus lead to chemoresistance. Med12 The deficiency has the effect of reshaping the immune microenvironment. Subsequent experiments have fully verified that the loss of MED12 in pancreatic cancer tumor cells can effectively promote CD8 + T cell infiltration and enhanced responsiveness of immune checkpoint inhibitors in pancreatic cancer significantly prolonged the survival of patients.

[0006] Human MED12 consists of 2177 amino acids, and we have demonstrated that MED12 expression in pancreatic cancer ductal cells is highly variable. MED12 The gene is located on the positive strand of human chromosome X, and its site information is as follows; Homo sapiens Chromosome X, Assembly GRCh38.p14, NC_000023.11: its transcript (NM_005120.3) (mRNA) sequence is shown in SEQ ID NO.1; its protein amino acid sequence is shown in SEQ ID NO.2.

[0007] As one of the three major components of heterochromatin proteins, human heterochromatin protein HP1A maintains the stability of heterochromatin by anchoring H3K9me2 / 3 on heterochromatin. Cbx5 After the deletion of HP1A, Med12 HP1A is composed of 191 amino acids, and we have fully demonstrated that MED12 inhibits retroviral activation by stabilizing HP1A. CBX5 The gene (encoding HP1A) is located on the antisense strand of human chromosome 12, and its site information is as follows; Homosapiens Chromosome 12, Assembly GRCh38.p14, NC_000012.12: its transcript (NM_001127321.1) (mRNA) sequence is shown in SEQ NO.3; its amino acid sequence is shown in SEQ ID NO.4. Summary of the invention

[0008] In view of the above technical problems in the prior art, the present invention provides the use of MED12 as an immunotherapy target in screening drugs for treating pancreatic cancer. The application is to solve the technical problem that the drugs in the prior art are not effective in treating pancreatic cancer.

[0009] The present invention also provides the use of MED12 as an immunotherapy target in screening drugs for treating pancreatic cancer, and the amino acid sequence of MED12 is shown in SEQ ID NO.2.

[0010] Furthermore, the immunotherapy of MED12 is achieved by regulating HP1A.

[0011] The present invention also provides the use of an immunotherapy drug or a chemotherapy drug combined with a drug that targets and inhibits MED12 in the preparation of a drug for treating pancreatic cancer, wherein the amino acid sequence of MED12 is shown in SEQ ID NO.2.

[0012] Furthermore, the immunotherapy drug is a PD-1 monoclonal antibody.

[0013] The present invention also provides the use of MED12 as a biomarker for screening targets for immunotherapy in the treatment of pancreatic cancer.

[0014] The present invention also provides the use of MED12 as a biomarker for screening targets for immunotherapy in the treatment of pancreatic cancer in the preparation of a detection kit.

[0015] The present invention also provides a detection kit, characterized in that it contains a reagent for detecting MED12. If MED12 is lowly expressed, the patient responds better to the immunosuppressant.

[0016] The present invention also provides use of a drug for targeted inhibition of MED12 in the preparation of a drug for treating pancreatic cancer, wherein the amino acid sequence of the MED12 is shown in SEQ ID NO.2.

[0017] The present invention also provides the use of MED12 in preparing a screening index for the treatment of pancreatic cancer. If MED12 is lowly expressed, the patient will respond better to the immunosuppressant.

[0018] The present invention provides a basis for screening the use of MED12, one of the main components of RNA polymerase II, in pancreatic cancer patients. MED12 has a high degree of homology in humans and mice. The present invention confirms that it is missing in pancreatic cancer cells in humans and mice. Med12 Can enhance the immunogenicity of pancreatic cancer cells.

[0019] In vivo experiments in mice showed that Med12 More tumor-killing T cells can be infiltrated into the missing tumors, and this group of T cells has stronger cytokine secretion and proliferation capabilities. Med12 The absence of can effectively promote the inhibitory effect of PD-1 monoclonal antibodies on tumors and significantly prolong the survival of tumor-bearing mice.

[0020] In a retrospective clinical study, it was found that in patients who had not received immunotherapy, the CD8 + More T cells; in patients treated with chemotherapy and PD-1 monoclonal antibodies, it was found that patients who responded better to the inhibitors generally had low expression of MED12, and patients with low MED12 expression had longer survival.

[0021] The function of MED12 is highly consistent in humans and mice. These data indicate that MED12 can be used as a screening indicator to guide the application of immunosuppressants in pancreatic cancer. At the same time, targeting MED12 is also expected to enhance the efficacy of immunotherapy such as ICB (Immune Checkpoint Blockade). MED12 can be used as a screening indicator to guide the application of immunosuppressants in pancreatic cancer. At the same time, targeting MED12 is also expected to enhance the efficacy of immunotherapy such as ICB.

[0022] Experiments have shown that in mouse models, the loss of MED12 in tumor cells can effectively promote CD8 + T cells accumulate in the tumor microenvironment, and this group of CD8 + The tumor killing function and proliferation ability of T cells were significantly improved. After treatment with PD-1 monoclonal antibody, Med12 The defective tumors can be effectively inhibited, and the survival time of these tumor-bearing mice is significantly prolonged.

[0023] The present invention provides a new target for immunotherapy of pancreatic cancer. Mechanism studies have shown that Med12 After deletion, the protein level of heterochromatin protein 1A (HP1A) decreases, thereby weakening the inhibitory effect of H3K9me3, further promoting the transcription level of retrovirus, activating retroviral elements, and ultimately mediating the activation of I / III interferon pathways. It promotes the expression of MHC-I, an antigen presenting molecule on the surface of tumor cells, thereby increasing CD8 + The recognition and killing of tumors by T cells is also due to CD8 + The accumulation of T cells in the tumor microenvironment enhances the effectiveness of PD-1 monoclonal antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 for Med12 Defective tumor cells reshape the immune microenvironment.

[0025] Figure 2 for Med12 The deficiency activates the type I / III interferon signaling pathway and promotes the expression of MHC-I.

[0026] Figure 3The interaction between MED12 and HP1A and the loss of HP1A promote type I / III interferon activation and upregulation of MHC-I.

[0027] Figure 4 Patients with low MED12 expression had more CD8 + T cells.

[0028] Figure 5 Patients with low MED12 expression are more responsive to PD-1 monoclonal antibodies and have longer survival.

[0029] Figure 6 This is a schematic diagram of the specific mechanism of action of MED12 in pancreatic cancer tumor cells.

[0030] The following are specific embodiments of the present invention, which are used to describe the present invention rather than to limit the present invention.

[0031] The reagents and experimental methods used in the examples are as follows:

[0032] Molecular biochemical reagents: cell lysis buffer (RIPA), protein immunoblotting antibody MED12 were purchased from Novus, antibodies MDA5 CGAS, STING, p-IRF3, IRF7, IRF9, P65, p-P65, STAT1, STAT3, p-STAT3 were purchased from CST, and chemiluminescent solution was purchased from Yamei. Flow cytometry antibodies CD45.2-AF700, CD4-BV650, CD8-PE-CY7, H2-Kb / Db-AF647, Granzyme B-PE, IFNg-BV421 were purchased from Biolegend, and TCF1-AF647 was purchased from CST. The flow cytometer used was purchased from BD. DAB colorimetric solution was purchased from Maixin.

[0033] Example 1

[0034] KPC1199 wild type was derived from the KPC spontaneous pancreatic tumor mouse model (purchased from Jackson), that is, pancreatic tissue was obtained from 12-15 week old KPC mice, primary cells were obtained by collagenase dissociation, and then epithelial cells were sorted and cultured on the wall.

[0035] Mouse pancreatic cancer cell line Med12 The knockout type is obtained by knocking out the Med12 gene through the CRISPR-Cas9 system based on the wild type KPC1199.

[0036] The mouse pancreatic cancer cell line KPC1199 wild type and Med12The knockout type was hit on both sides of the mouse groin with 1 million cells. The mice were killed 14 days later to remove the tumor. The tumor was cut into pieces with ophthalmic scissors, placed in a digestion solution containing 2mg / ml IV collagenase, and placed in a 37-degree water bath for digestion for 30 minutes. Every 10 minutes, it was taken out and placed on an oscillator for 15 seconds. When the digestion was completed, an equal volume of HBSS buffer was added to terminate the digestion. The filtrate was collected by filtering with a 200-mesh filter at 1000 rpm, centrifuged for 5 minutes, and the supernatant was discarded. After resuspending with complete medium, a final concentration of 100ng / ml phorbol ester, 1µg / ml ionomycin, and 5µg / ml brefeldin A were added, and the cells were cultured in a 37°C cell culture incubator for 4 hours. After 4 hours of cell stimulation, the cells were collected, added with 1ml / tube HBSS, gently mixed, centrifuged at 400-600g, 5min, and the supernatant was discarded and gently mixed. Resuspend with 100µl / tube HBSS, add the required flow cytometry antibodies CD45.2-AF700, CD4-BV650, CD8-PE-CY7 premixed into cocktail (1:200, 0.5µl / ab / tube) to the flow cytometry tube. Incubate at 4℃ in the dark for 30 minutes. After incubation, add 1ml / tube HBSS, gently mix, 400-600g, 5min centrifuge. After discarding the supernatant, add 400µl / tube fixed membrane permeabilization agent, blow it away with a gun, and incubate at 4℃ in the dark for 30 minutes. After the membrane is permeabilized, add 1ml / tube washing solution, blow it away at 400-600g, 5min centrifuge, and discard the supernatant. Intracellular staining: Add the intracellular antibodies GranzymeB-PE and IFNg-BV421 to the detergent of 100µl / tube system at a ratio of 1:200, mix well, add 100µl / tube to the flow tube, and incubate at 4 degrees in the dark for 30 minutes. After the intracellular staining is completed, add +1ml / tube of washing solution, mix well, centrifuge at 400-600g for 5min, and discard the supernatant. Resuspend and fix with 2% paraformaldehyde and use flow cytometer to collect data.

[0037] Figure 1 The mouse pancreatic cancer cell line KPC1199 wild type and Med12 The number of CD4 and CD8 infiltrating knockout subcutaneous tumors (A) and CD8 + Expression ratio of T cell functional factors (B).

[0038] Figure 1 The data show that Med12 The number of CD8+T cells in knockout tumors increased significantly and their function was enhanced.

[0039] Example 2

[0040] Mouse pancreatic cancer cell lines KPC1199 wild type and Med12Knockout cell lines, 37 degrees incubator, 3 minutes, complete medium to terminate digestion, pipetting and mixing, collect cells in 15ml centrifuge tube, 1000rpm, 5 minutes centrifugation. Add 800µl cell lysis buffer to each cell, add PMSF protease inhibitor and protease phosphatase inhibitor 1X at a final concentration of 1mM to the cell lysis buffer, pipetting and mixing, place on ice for 45 minutes, high-speed centrifugation at 12000rpm, 15 minutes, take the supernatant lysis buffer and place in a new EP tube, quantify with BCA protein quantitative detection kit and balance, add SDS-PAGE at a final concentration of 1X at 100 degrees, denature for 10 minutes, and perform protein immunoblotting. After loading, wait until the protein marker runs to a position about 1cm away from the lower edge of the separation gel, and stop. Transfer the membrane with 20% methanol transfer solution for 1.5 hours, place the transfer tank in an ice-water mixture, and block with 5% skim milk at room temperature for 1 hour after transfer. Take the PVDF membrane of the corresponding band and incubate the primary antibody at 4°C overnight. Take it out the next day and wash it 3 times with TBST on a horizontal shaker at room temperature for 10 minutes each time. After the last TBST wash, discard it, add the secondary antibody containing horseradish peroxidase HRP and incubate it at room temperature for 1 hour, and then wash it 3 times with TBST on a horizontal shaker for 10 minutes each time. Develop and collect data.

[0041] Figure 2 A and 2 B show the type I / III interferon signaling pathway in Med12 It is activated in knockout cell lines, thereby promoting the expression of MHC-I molecules on the surface of tumor cells ( Figure 2 C).

[0042] Figure 3 A is Med12 The protein level of HP1A before and after knockout. The results showed Med12 HP1A protein levels dropped dramatically after deletion. Figure 3 B and Figure 3 C is the immunoprecipitation of MED12 and HP1A, and the results show that MED12 and HP1A interact with each other.

[0043] Figure 3 The DE results showed that Cbx5 After deletion, H3K9me3 is downregulated, type I / III interferon pathway is activated, and MHC-I molecules are upregulated.

[0044] Example 3

[0045] We collected two sets of surgical resection or tumor puncture samples from pancreatic cancer patients to verify the function of MED12. Pancreatic cancer specimens are wax block tissues. The tissue sections were first placed in a 55-degree oven to dissolve for 30 minutes, then placed in xylene and different concentration gradients of ethanol for step-by-step dewaxing, and then blocked with 5% goat serum at room temperature for one hour, and then incubated with primary antibodies at 4°C overnight. The wet box was removed the next day, and after returning to room temperature, it was washed 3 times with PBST on a horizontal shaker for 5 minutes each time to wash away the unbound primary antibody. After incubating the secondary antibody at room temperature for 1 hour, the DAB color development time was controlled according to the expression level of the detected protein. Subsequently, the slides were sealed with resin and scanned to collect data.

[0046] Figure 4 For patients who have not received immunotherapy, the number of CD8 infiltrating CD8 cells around the adjacent tissues of the cancer was counted according to the expression of MED12. + The number of T cells showed that patients with low MED12 expression had CD8 + There is more T cell infiltration (A is the MED12 expression gradient, B is the CD8a expression gradient, and C is the number of CD8 in tumors with different MED12 expression levels).

[0047] Figure 5 These are samples from patients who underwent combined chemotherapy and immunotherapy. The results showed that MED12 also showed different expression levels in these patients (A). Patients who responded better to immunotherapy generally had low expression of MED12 (B), and patients with low expression of MED12 had significantly prolonged survival (C).

Claims

1. The use of MED12 as an immunotherapy target in screening drugs for treating pancreatic cancer, characterized in that: The amino acid sequence of MED12 is shown in SEQ ID NO.

2. When MED12 is lowly expressed, the patient responds better to immunosuppressants. MED12 is used as an immunotherapy target to treat pancreatic cancer by regulating HP1A, and the immunotherapy is achieved by PD-1 monoclonal antibody.

2. Use of MED12 as a biomarker in the preparation of a reagent for screening a population for immunotherapy of pancreatic cancer, characterized in that: When MED12 is lowly expressed, the patient responds better to immunosuppressants. MED12 is used as an immunotherapy target to treat pancreatic cancer by regulating HP1A, and the immunotherapy is achieved by PD-1 monoclonal antibodies.

3. Use of a drug that targets and inhibits MED12 in the preparation of a drug for treating pancreatic cancer, characterized in that: The amino acid sequence of MED12 is shown in SEQ ID NO.

2. When MED12 is lowly expressed, the patient responds better to immunosuppressants. MED12 is used as an immunotherapy target to treat pancreatic cancer by regulating HP1A, and the immunotherapy is achieved by PD-1 monoclonal antibody.

4. Use of MED12 as a screening index in the preparation of a reagent for guiding immunotherapy of pancreatic cancer, characterized in that: When MED12 is lowly expressed, the patient responds better to immunosuppressants. MED12 is used as an immunotherapy target to treat pancreatic cancer by regulating HP1A, and the immunotherapy is achieved by PD-1 monoclonal antibodies.

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