Test mixtures to predict sensitivity of tumor to prmt5 inhibitor treatment
By detecting BRCA gene mutations, DNA damage, and IFN pathway levels, a reagent was prepared to predict the sensitivity of tumors to PRMT5 inhibitor therapy, solving the problem of difficulty in predicting tumor sensitivity in existing technologies and achieving personalized treatment effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ZHEJIANG CANCER HOSPITAL
- Filing Date
- 2023-04-21
- Publication Date
- 2026-05-01
AI Technical Summary
Existing technologies are insufficient to effectively predict the sensitivity of tumors to PRMT5 inhibitors, and there is a lack of accurate biomarkers for personalized treatment.
By detecting BRCA gene mutations, DNA damage levels, and IFN pathway levels, reagents for predicting the sensitivity of tumors to PRMT5 inhibitor therapy were prepared using BRCA gene mutation detection reagents, DNA damage level detection reagents, and IFN pathway level detection reagents.
Accurately predicting patients' sensitivity to PRMT5 inhibitors can improve treatment effectiveness and enable personalized treatment.
Smart Images
Figure CN116356032B_ABST
Abstract
Description
A mixture of assays to predict the sensitivity of tumors to PRMT5 inhibitor therapy. Technical Field
[0001] This invention relates to the field of biomedical technology, and more specifically to a detection mixture for predicting the sensitivity of tumors to PRMT5 inhibitor therapy. Background Technology
[0002] PRMT5 (arginine methyltransferase 5) is highly expressed in various tumors, and its expression level is closely related to tumor occurrence, development, and prognosis. PRMT5 plays an important role in various cellular life processes, including RNA splicing, transcription, DNA repair, signal transduction, cell proliferation, cell differentiation, apoptosis, and tumorigenesis. PRMT5 participates in the assembly of spliceosomes, Golgi apparatus, and 40S ribosomes, promoting tumor development and progression. Therefore, PRMT5 inhibitors have potential anti-tumor effects. Currently, there are 26 PRMT5 inhibitors under investigation, most of which are in early clinical trials, with GSK3326595 currently in Phase II clinical trials being the most advanced. Identifying biomarkers that can predict patient sensitivity to PRMT5 inhibitors is crucial for achieving precision medicine using PRMT5 inhibitors. BRCA1 / 2 genes play an important role in regulating human cell replication, DNA damage repair, and normal cell growth. When cells suffer DNA damage, especially double-stranded breaks (DSBs), focal points formed by γH2AX appear at the DSB sites. γH2AX is a specific indicator for detecting cellular DNA damage. The IFN (interferon) pathway is a potent cytokine and key component of the first line of defense against viral infection, primarily controlling inflammation and immunity by directly inducing antipathogen molecular responses that inhibit viral replication. This invention discovers that BRCA gene mutation status, DNA damage levels, and IFN pathway expression levels are biomarkers that can predict PRMT5 inhibitor sensitivity in triple-negative breast cancer. Stratifying patient treatment based on these biomarkers may improve the efficacy of PRMT5 inhibitors. Summary of the Invention
[0003] By correlating drug sensitivity with gene mutation status and transcriptome sequencing data, it was found that BRCA gene mutation status, DNA damage, and IFN pathway expression levels can serve as biomarkers for predicting PRMT5 inhibitor sensitivity. Therefore, the present invention aims to provide a biomarker for predicting the sensitivity of tumors to PRMT5 small molecule inhibitors, comprising: BRCA gene mutation, DNA damage level, and IFN pathway level.
[0004] To achieve the above-mentioned objectives, the following technical solution is adopted.
[0005] The use of the assay mixture in the preparation of reagents for predicting the sensitivity of PRMT5 inhibitors to the prevention and / or treatment of solid tumors, wherein the assay mixture comprises at least one of the following:
[0006] BRCA gene mutation detection reagent; and / or,
[0007] DNA damage level detection reagent; and / or,
[0008] IFN pathway level detection agent.
[0009] Preferably, BRCA gene mutations include BRCA1 and BRCA2 genes.
[0010] This invention experimentally verified that most cell lines sensitive to PRMT5 inhibitors have BRCA1 or BRCA2 gene mutations. Therefore, BRCA gene mutations can serve as biomarkers for predicting PRMT5 inhibitor sensitivity. Consequently, BRCA gene mutation detection reagents can be used to prepare agents for predicting the sensitivity of solid tumors to PRMT5 inhibitor prevention and / or treatment.
[0011] Preferably, the BRCA gene mutation detection agent includes a BRCA mutation gene detection kit.
[0012] More preferably, the BRCA mutation gene detection kit includes a BRCA1 and / or BRCA2 mutation gene detection kit.
[0013] Preferably, the level of DNA damage includes the γH2AX level.
[0014] This invention experimentally verifies that γH2AX expression is high in cell lines sensitive to PRMT5 inhibitors, while γH2AX is almost not expressed in insensitive cell lines. Therefore, DNA damage level can be used to predict the sensitivity of PRMT5 inhibitors for the prevention and / or treatment of solid tumors. Thus, DNA damage level detection reagents can be used to prepare reagents for predicting the sensitivity of PRMT5 inhibitors for the prevention and / or treatment of solid tumors.
[0015] Preferably, the DNA damage level detection agent includes a DNA damage detection kit.
[0016] More preferably, the DNA damage detection kit includes a γH2AX detection kit.
[0017] Preferably, the IFN pathway level includes IFN expression level.
[0018] Preferably, the IFN pathway includes IFN-α and IFN-γ.
[0019] This invention, through GSEA pathway analysis, discovered that IFN pathway levels are correlated with the sensitivity of solid tumors to PRMT5 inhibitors during treatment. Therefore, IFN pathway levels can be used to predict the sensitivity of solid tumors to PRMT5 inhibitors for prevention and / or treatment. Consequently, IFN pathway level detection reagents can be used to prepare reagents for predicting the sensitivity of solid tumors to PRMT5 inhibitors for prevention and / or treatment.
[0020] Preferably, the IFN pathway level detection agent includes an IFN detection kit.
[0021] More preferably, the IFN detection kit includes an IFN-α and / or IFN-γ detection kit.
[0022] Preferably, the aforementioned solid tumors include benign solid tumors and malignant solid tumors.
[0023] More preferably, benign solid tumors include: hamartomas, leiomyomas, hemangiomas, lymphangiomas, as well as various adenomas and adenomatous polyps.
[0024] More preferably, malignant solid tumors include: Hodgkin's lymphoma, non-Hodgkin's lymphoma, lung cancer, breast cancer, ovarian cancer, gastric cancer, colon cancer, rectal cancer, liver cancer, pancreatic cancer, head and neck malignant tumors, urinary system malignant tumors, endometrial cancer, cervical cancer, osteosarcoma, chondrosarcoma, Ewing's sarcoma, thyroid cancer, hepatoblastoma, and nephroblastoma.
[0025] More preferably, solid tumors include triple-negative breast cancer.
[0026] More preferably, triple-negative breast cancer includes the following cell lines:
[0027] Hs578T, SUM-159-PT, HCC3153, MDAMB436, CAL-120, SUM-149-PT, MDAMB231, BT549, MB157, HCC70, MX1, HCC38, HCC1395, HCC1143, HCC1806 or MDAMB468.
[0028] More preferably, triple-negative breast cancer includes the following cell lines:
[0029] Hs578T, SUM-159-PT, HCC1806 or MDAMB468.
[0030] Preferably, the PRMT5 inhibitor is selected from GSK3326595, AMG-193, MRTX1719, TNG-908, PF-069399999, PRT543, PRT811, SH-3765, Onametostat, SCR-6920, SKL27969, SYHX2001, AGX323, BRD0639, C220, DS-437, DW14800, GSK3203591, GSK3235025, JBI-778, LLY-283, MRTX9768, MS4322, MS4369, MS4370, or PF-06855800.
[0031] More preferably, the PRMT5 inhibitor is selected from LLY-283 or GSK591.
[0032] More preferably, the dose range of the PRMT5 inhibitor is selected from 10-100 mg, for example, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, or 100 mg.
[0033] More preferably, the PRMT5 inhibitor can be administered via oral, parenteral, or transdermal routes.
[0034] More preferably, parenteral administration includes, but is not limited to, intravenous injection, subcutaneous injection, and intramuscular injection.
[0035] More preferably, the PRMT5 inhibitor is administered once daily, twice daily, three times daily, once weekly, once every two weeks, once every three weeks, or once monthly.
[0036] This invention also discloses a detection mixture for predicting the sensitivity of tumors to PRMT5 inhibitor therapy, comprising:
[0037] BRCA gene mutation detection reagent; and / or,
[0038] DNA damage level detection reagent; and / or,
[0039] IFN pathway level detection agent.
[0040] Preferably, the PRMT5 inhibitor is selected from GSK3326595, AMG-193, MRTX1719, TNG-908, PF-069399999, PRT543, PRT811, SH-3765, Onametostat, SCR-6920, SKL27969, SYHX2001, AGX323, BRD0639, C220, DS-437, DW14800, GSK3203591, GSK3235025, JBI-778, LLY-283, MRTX9768, MS4322, MS4369, MS4370, or PF-06855800.
[0041] This invention discloses the use of BRCA gene mutation detection agents in the preparation of reagents for predicting the sensitivity of PRMT5 inhibitors to the prevention and / or treatment of solid tumors.
[0042] Preferably, BRCA gene mutations include BRCA1 and BRCA2 genes.
[0043] Preferably, the BRCA gene mutation detection agent includes a BRCA mutation gene detection kit.
[0044] More preferably, the BRCA mutation gene detection kit includes a BRCA1 and / or BRCA2 mutation gene detection kit.
[0045] The present invention also discloses the use of DNA damage level detection reagents in the preparation of reagents for predicting the sensitivity of PRMT5 inhibitors to prevent and / or treat solid tumors.
[0046] Preferably, the level of DNA damage includes the γH2AX level.
[0047] Preferably, the DNA damage level detection agent includes a DNA damage detection kit.
[0048] More preferably, the DNA damage detection kit includes a γH2AX detection kit.
[0049] The present invention also discloses the use of IFN pathway level detection agents in the preparation of reagents for predicting the sensitivity of PRMT5 inhibitors to prevent and / or treat solid tumors.
[0050] Preferably, the IFN pathway level includes IFN expression level.
[0051] Preferably, the IFN pathway includes IFN-α and IFN-γ.
[0052] Preferably, the IFN pathway level detection agent includes an IFN detection kit.
[0053] More preferably, the IFN detection kit includes an IFN-α and / or IFN-γ detection kit.
[0054] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0055] This invention experimentally verified that most PRMT5 inhibitor-sensitive cell lines exhibit BRCA1 or BRCA2 gene mutations, high γH2AX expression levels, or high IFN expression levels. This demonstrates that BRCA gene mutations, DNA damage levels, or IFN pathway levels can serve as biomarkers for predicting PRMT5 inhibitor sensitivity. Therefore, BRCA gene mutation detection agents, DNA damage level detection agents, or IFN pathway level detection agents can be used to prepare reagents for predicting the sensitivity of patients to PRMT5 inhibitors in the prevention and / or treatment of solid tumors. BRCA gene mutations, DNA damage levels, or IFN pathway levels can effectively and accurately predict a patient's sensitivity to PRMT5 inhibitors, thereby facilitating personalized treatment and increasing treatment effectiveness. Attached Figure Description
[0056] Figure 1 shows the inhibitory effect of the PRMT5 inhibitor LLY283 on the TNBC cell line;
[0057] Figure 2 shows the inhibitory effect of the PRMT5 inhibitor GSK591 on the TNBC cell line;
[0058] Figure 3 shows the predicted mutation landscape of the PRMT5 inhibitor LLY283;
[0059] Figure 4 shows the predicted mutation landscape of the PRMT5 inhibitor GSK591;
[0060] Figure 5 shows the immunofluorescence staining of γH2AX in sensitive and insensitive cell lines;
[0061] Figure 6 shows the γH2AX levels in sensitive and insensitive cell lines as displayed by Western blot.
[0062] Figure 7 shows the GSEA analysis of the HALLMARK pathway related to PRMT5 inhibitor sensitivity;
[0063] Figure 8 shows the GSEA diagram of the interferon pathway. Detailed Implementation
[0064] Exemplary embodiments will now be described in detail. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this disclosure. Rather, they are merely examples of methods consistent with some aspects of this disclosure.
[0065] Unless otherwise specified, the experimental methods used in the following examples are conventional methods or are performed according to the conditions recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0066] Example 1
[0067] Validating BRCA gene mutations as a biomarker for predicting PRMT5 inhibitors
[0068] I. Inhibitory effect of PRMT5 inhibitors on TNBC cell line
[0069] 1. Test drug
[0070] Drug names: PRMT5 inhibitor LLY-283, GSK591;
[0071] 2. Cell lines
[0072] Cell line names: Hs578T, SUM-159-PT, HCC3153, MDAMB436, CAL-120, SUM-149-PT, MDAMB231, BT549, MB157, HCC70, MX1, HCC38, HCC1395, HCC1143, HCC1806, MDAMB468;
[0073] 3. Breeding environment: Temperature: Controlled temperature 37℃; 5% CO2 incubator;
[0074] 4. Culture medium: high glucose DMEM, 10% fetal bovine serum, antibiotics;
[0075] RPMI 1640, 10% fetal bovine serum, penicillin antibody;
[0076] Experimental steps:
[0077] The TNBC cell line described above was seeded into 384-well plates with 3000 cells per well. After cell adhesion, a concentration gradient drug treatment was set up, starting with 10 μM and diluting by half, with 8 concentration gradients. DMSO was used as a control group. After 5 days of treatment, cell coverage was measured using Incucyte. The area on curve (AAC) was calculated, and the results are shown in Figures 1 and 2.
[0078] As shown in Figures 1 and 2, different cell lines have different sensitivities to PRMT5 inhibitors. The sensitivity of each cell line was recorded for subsequent experiments.
[0079] II. Validating BRCA gene mutation as a biomarker for predicting PRMT5 inhibitors
[0080] 1. Test drug
[0081] Drug names: PRMT5 inhibitors LLY283 and GSK591;
[0082] 2. Mutation Analysis
[0083] Cell line gene mutation information is from Cancer Cell Line Encyclopedia (CCLE);
[0084] Cell lines were sorted according to their sensitivity to PRMT5 inhibitors and their mutation information (common gene mutations in triple-negative breast cancer) was mapped to the Cancer Cell Line Encyclopedia (CCLE). The mutation landscape is shown in Figures 3 and 4. Figures 3 and 4 show that most PRMT5 inhibitor-sensitive cell lines have BRCA1 / 2 gene mutations. Therefore, BRCA gene mutations can serve as biomarkers for predicting PRMT5 inhibitor sensitivity. Thus, BRCA gene mutation detection reagents can be used to prepare agents for predicting the sensitivity of solid tumors to PRMT5 inhibitors for prevention and / or treatment.
[0085] Example 2
[0086] Validating DNA damage levels as a biomarker for predicting PRMT5 inhibitors
[0087] 1. Test drug
[0088] Drug Name: PRMT5 inhibitor LLY283, GSK591
[0089] 2. Cell lines
[0090] The following cell lines were used to conduct experiments to detect DNA damage in the cell lines:
[0091] Hs578T, SUM-159-PT, HCC1806, MDAMB468;
[0092] Among them, the sensitive cell lines are HCC1806 and MDAMB468; the insensitive cell lines are Hs578T and SUM-159-PT.
[0093] 3. Breeding environment: Temperature: Controlled temperature 37℃; 5% CO2 incubator
[0094] 4. Culture medium: high glucose DMEM, 10% fetal bovine serum, antibiotics
[0095] RPMI 1640, 10% fetal bovine serum, penicillin antibody
[0096] 5. Measure the level of cellular DNA damage
[0097] The level of cellular DNA damage was determined using Western blot and immunofluorescence, respectively, with the experimental procedures as follows:
[0098] Immunofluorescence:
[0099] (1) MDAMB468, HCC1806, SUM159PT and Hs578T cells were plated into confocal dishes, the culture medium was aspirated, and the cells were washed twice with PBS for 1 min each time. During this time, the dishes were gently shaken 3-5 times.
[0100] (2) After discarding the PBS in the small dish and well, add 1 ml of 4% paraformaldehyde fixative (prepared with PBS at pH 7.4) to fix the cells for 10 min;
[0101] (3) Discard the paraformaldehyde fixative and wash the cells twice with PBS for 1 min each time;
[0102] (4) After discarding the PBS in the small dish and well, add 0.2% Trion X-100 prepared with PBS and permeate for 8-15 min (depending on the density of the cells). Then wash the cells twice with PBS, 1 min each time.
[0103] (5) Block the cells with 3% BSA prepared with PBS for 30 min;
[0104] (6) Dilute the antibody with 3% BSA to a final volume of 200 μl. The antibody dilution ratio is 1:500.
[0105] (7) Discard the blocking solution in the small dish and the round well, and add antibody immunolabeling for 1-2 hours;
[0106] (8) Wash the cells twice with PBS, 10 min each time;
[0107] (9) After discarding the PBS in the round wells, the secondary antibody conjugated with fluorescein was diluted with 3% BSA at a ratio of 1:200-500, with a final volume of 200 μl, and Sf9 cells were incubated in the dark for 60 min.
[0108] (10) Wash the cells twice with PBS, 10 min each time;
[0109] (11) Stain with DAPI at room temperature for 3-5 min, wash with PBS 3 times for 3-5 min each time, and mount with anti-fluorescence quenching mounting solution;
[0110] (12) Observe and photograph using a Leica TCS SP5 laser confocal microscope.
[0111] Western blot:
[0112] (1) Extraction of MDAMB468, HCC1806, SUM159PT, and Hs578T cell proteins;
[0113] (2) BCA quantification;
[0114] (3) Preparation of protein gel (SDS-PAGE gel);
[0115] (4) Protein sample denaturation;
[0116] (5) Electrophoresis;
[0117] (6) Transfer membrane;
[0118] (7) Closed;
[0119] (8) Primary antibody;
[0120] (9) TBST washing;
[0121] (10) Secondary antibody;
[0122] (11) TBST washing;
[0123] (12) Development;
[0124] The above experimental procedures were all performed according to standard experimental procedures and will not be described in detail here. The results of immunofluorescence are shown in Figure 5, and the results of Western blot are shown in Figure 6.
[0125] As shown in Figure 5, the amount of fluorescence in sensitive cells was significantly higher than that in insensitive cells, while almost no fluorescence was observed in insensitive cells. This indicates that the expression level of γH2AX is high in sensitive cells, which corresponds to a higher level of DNA damage; therefore, the level of DNA damage can be used to predict the effectiveness of PRMT5 inhibitors.
[0126] As shown in Figure 6, the expression level of γH2AX is high in sensitive cell lines and almost non-sensitive cell lines. The presence of a large amount of γH2AX expression in sensitive cells indicates a high level of DNA damage, which is consistent with the conclusion obtained from immunofluorescence staining. This suggests that the level of DNA damage can be used to predict the effect of PRMT5 inhibitors.
[0127] Example 3
[0128] Identifying IFN pathway expression levels as biomarkers for predicting PRMT5 inhibitors
[0129] 1. Test drug
[0130] Drug names: PRMT5 inhibitor LLY-283, GSK591;
[0131] 2. Cell lines
[0132] Cell line names: Hs578T, SUM-159-PT, HCC3153, MDAMB436, CAL-120, SUM-149-PT, MDAMB231, BT549, MB157, HCC70, MX1, HCC38, HCC1395, HCC1143, HCC1806, MDAMB468;
[0133] 3. Breeding environment: Temperature controlled at 37℃; 5% CO2 incubator;
[0134] 4. Culture medium: high glucose DMEM, 10% fetal bovine serum, antibiotics;
[0135] RPMI 1640, 10% fetal bovine serum, penicillin antibody;
[0136] Plotting steps: Perform correlation analysis between the above cell line RNA-seq data and PRMT5 inhibitor sensitivity to obtain the correlation coefficient and significance p-value of each gene and sensitivity expression, and perform GSEA pathway analysis.
[0137] The GSEA pathway analysis results are shown in Figures 7 and 8. Figure 7 shows that IFN-α and IFN-γ are associated with the PRMT5 inhibitors LLY-283 and GSK591, and Figure 8 further validates this result. This indicates that IFN expression can be used to predict the efficacy of PRMT5 inhibitors.
[0138] Experimental Example 1
[0139] Predicting the sensitivity of tumors to PRMT5 inhibitor therapy
[0140] 1. Cell lines
[0141] Tumor tissues from 18 patients diagnosed with triple-negative breast cancer were cultured and used as samples. BRCA gene mutation detection, DNA damage level detection, and IFN pathway detection were performed according to the instructions of the corresponding kits. During the detection, simultaneous mutations in BRCA1 and BRCA2 were recorded as insensitive; significantly elevated γH2AX levels (using HCC1806 as a control in this case, compared to known PRMT5 inhibitor-sensitive cell lines) were recorded as insensitive; and high expression of IFN-α and IFN-γ (using HCC1806 as a control in this case, compared to known PRMT5 inhibitor-sensitive cell lines) were recorded as insensitive.
[0142] 2. Breeding environment: Temperature controlled at 37℃; 5% CO2 incubator;
[0143] 3. Culture medium: high glucose DMEM, 10% fetal bovine serum, antibiotics;
[0144] 4. Reagent Kit: Human BRCA1 and BRCA2 Gene Mutation Detection Kit (Reversible Termination Sequencing Method) (Xiamen AmoyDx Biotechnology Co., Ltd.);
[0145] DNA Damage Detection Kit (γ-H2AX Immunofluorescence Assay) (Beyotime Biotechnology Co., Ltd.);
[0146] Human IFN-γ ELISA kit, Human IFN-α ELISA kit (Beyotime Biotechnology Co., Ltd.);
[0147] The test results are shown in Table 1.
[0148] Table 1. Statistics on the sensitivity of patient tumor tissue to PRMT5 inhibitors
[0149] Number of sensitive samples (pieces) Number of insensitive samples (pieces) BRCA gene mutation 135 DNA damage level 135 IFN pathway detection 135 surface
[0150] Table 1 shows that the results predicted by the three detection methods were consistent. Subsequent follow-up visits revealed that 13 patients were PRMT5 inhibitor-sensitive, and 5 were PRMT5 inhibitor-insensitive, consistent with the predicted results. Therefore, BRCA gene mutations, DNA damage levels, or IFN pathway levels can serve as biomarkers for predicting PRMT5 inhibitor sensitivity. Furthermore, BRCA gene mutation detection agents, DNA damage level detection agents, or IFN pathway level detection agents can be used to prepare reagents for predicting the sensitivity of PRMT5 inhibitors in the prevention and / or treatment of solid tumors.
[0151] The conventional operations in the operation steps of this invention are well known to those skilled in the art and will not be described in detail here.
[0152] The embodiments described above provide a detailed explanation of the technical solution of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.
Claims
1. The use of a detection mixture in the preparation of a reagent for predicting the sensitivity of solid tumors to PRMT5 inhibitor therapy, characterized in that, The detection mixture includes: an IFN pathway level detection agent; the IFN pathway level detection agent includes an IFN detection kit, the IFN detection kit includes an IFN-α and / or IFN-γ detection kit; the solid tumor is triple-negative breast cancer; the PRMT5 inhibitor is selected from LLY-283 and GSK591.