Co-targeting DNA methylation-H3K27me3 for reversing prostate cancer castration resistance and immunosuppression

Abstract

The invention belongs to the technical field of cancer treatment, and discloses application of co-targeting EZH2 / DNMT to synergistically reversing castration-resistant prostate cancer resistance. We prove that dual targeting of DNMT and EZH2 breaks epigenetic transformation, so that tumors are re-sensitive to androgen deprivation therapy. The strategy not only eliminates CRPC in a preclinical model, but also reverses immunosuppression, so that cytotoxic T cell infiltration is increased by 11.4 times. We data extend the use of EZH2 inhibitors beyond PRC2 dependent cancers, localizing the interaction of DNA methyltransferase with EZH2 as a targeting weakness in extracellular matrix rich solid tumors. The combination therapy provides a mandatory solution to overcome stromal disorders, which provides a revolutionary strategy for biomechanical factor driven malignancies.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cancer treatment, and more particularly relates to co-targeting DNA methylation-H3K27me3 to reverse castration-resistant prostate cancer and immunosuppression. BACKGROUND

[0002] Prostate cancer (PCa) remains the leading cause of male-related cancer deaths worldwide, and castration-resistant prostate cancer (CRPC) occurs after androgen deprivation therapy (ADT) treatment. Despite advances in the treatment of prostate cancer targeting androgen receptor (AR) signaling pathways, drug resistance inevitably occurs through mechanisms such as AR splice variants, neuroendocrine differentiation, and epigenetic reprogramming. Notably, CRPC is associated with significant extracellular matrix (ECM) remodeling, which enhances tumor stiffness, activates non-AR-dependent survival pathways, and promotes the development of treatment resistance. During the development of CRPC, epigenetic changes regulate transcriptional activity and play an important role in the pro-cancer function of AR signaling. Although epigenetic disorders, particularly DNA hypermethylation and polymerase-mediated histone modification, are associated with cancer development, the interaction between these suppressive markers and their potential functional effects on extracellular matrix-driven castration-resistant prostate cancer (CRPC) remain unclear.

[0003] A key obstacle to achieving effective epigenetic therapy in solid tumors is the compensatory interaction between DNA methylation and H3K27me3. H3K27me3 is a histone modification deposited by polycomb repressive complex 2 (PRC2), which maintains a transcriptionally silent state. Drug inhibition of DNA methyltransferases (DNMTs) often fails to reactivate tumor suppressor genes (TSGs) because of the accumulation of H3K27me3 opposite to DNA methylation at sites previously silenced by DNA methylation, which is not yet understood in extracellular matrix (ECM)-rich solid tumors such as castration-resistant prostate cancer. This epigenetic plasticity resembles the bimodal nature of development, with a coexisting H3K4me3 and H3K27me3 "pre-set" state in CpG island promoters, but hijacked for stable repression in cancer.

[0004] The prior art lacks effective strategies to break the progression of castration-resistant prostate cancer and treatment resistance. SUMMARY

[0005] To overcome the deficiencies of the prior art, the present application aims to break the mutual epigenetic transformation relationship between H3K27me3 and DNA methylation, ultimately making CRPC sensitive to androgen deprivation therapy and reversing the resistance of castration-resistant prostate cancer.

[0006] The primary object of the present application is to provide a synergistic inhibition of CRPC by targeting and inhibiting EZH2 and DNMT.

[0007] The object of the present application is achieved by the following technical solutions.

[0008] The present application first found, through cancer genome atlas analysis, that the levels of EZH2, DNMT3A and DNMT3B in 497 cases of prostate cancer patients were significantly higher than those in 52 healthy individuals. In addition, high levels of EZH2, DNMT1, DNMT3A and DNMT3B expression levels were associated with shorter survival, indicating poor clinical prognosis of prostate cancer.

[0009] Therefore, the present application first provides the use of product A in the preparation of a kit or reagent for improving the accuracy of clinical diagnosis or prognosis of CRPC, wherein the product A comprises a substance for detecting the expression level of the EZH2 gene and / or the DNMT gene.

[0010] Next, the present application studies the interaction between H3K27me3 regulation and DNA methylation (5mC), and the results show that in CRPC, there is a bidirectional compensatory conversion between H3K27me3 and DNA methylation regulated by EZH2 and DNMT1, respectively.

[0011] Therefore, the present application also provides the use of the EZH2 / DNMT gene or its expression product in the development and screening of functional products for CRPC, wherein the functional product has an inhibitory effect on the EZH2 / DNMT gene or its expression product; and the functional product can break the compensatory epigenetic transformation driven by DNA methylation and H3K27me3 in CRPC.

[0012] On the basis of the above research content, the present application proposes a new treatment strategy, i.e. a synergistic inhibition of CRPC by simultaneously targeting EZH2 and DNMT.

[0013] Therefore, the present application also provides the use of the functional product having an inhibitory effect on the EZH2 / DNMT gene or its expression product in the preparation of a product for treating CRPC.

[0014] First, in vitro experiments showed that GSK126 (an EZH2 inhibitor) and DAC (a DNMTs inhibitor) combination treatment induced significantly higher apoptosis compared to single agent treatment. Consistently, combination treatment significantly inhibited cell viability and formed fewer cell colonies. In addition, GSK126 and DAC combination treatment significantly reduced cell migration compared to single agent treatment, indicating that the treated cells had reduced mobility. Furthermore, combination treatment significantly inhibited cell invasion. The near complete inhibition of invasion and migration indicates that metastatic ability was disrupted, which is a key feature of treatment refractory CRPC.

[0015] Next, the present application found that EZH2 knockdown alone only modestly reduced tumor volume, but when combined with the DNMT inhibitor decitabine, a synergistic effect on tumor growth was observed: the tumor volume in the combination treatment group was reduced by 90% compared to the control group. Similarly, the tumor weight in the combination treatment group was also significantly lower than that in the single agent treatment group.

[0016] Therefore, as a preferred mode of the above technical solution, the functional product that has an inhibitory effect on EZH2 / DNMT genes or their expression products is used in the preparation of a product for treating castration-resistant prostate cancer. When the functional product is used in vitro, the functional product has the following effects:

[0017] (1) promotes apoptosis of castration-resistant prostate cancer cells, and / or

[0018] (2) inhibits proliferation of castration-resistant prostate cancer cells, and / or

[0019] (3) inhibits migration of castration-resistant prostate cancer cells, and / or

[0020] (4) inhibits invasion of castration-resistant prostate cancer cells;

[0021] When the functional product is used in vivo, the functional product has the following effects:

[0022] (1) inhibits tumor growth, reduces tumor volume, and / or

[0023] (2) degrades tumor tissue extracellular matrix, reduces tumor stiffness.

[0024] To explore the immunomodulatory effect, a syngeneic subcutaneous model using EZH2 knockdown TRAMP-C1 cells was established in immunocompetent mice. H&E and immunofluorescence analysis results were consistent with the previous results, showing an 8.5-fold increase in apoptosis and a 35% inhibition of proliferation. Collagen degradation was further exacerbated in this model. Given the presence of an immunosuppressive tumor microenvironment (TME) in the tumor, characterized by a decrease in cytotoxic CD8+ T cells and an increase in CD206+ macrophages compared to normal tissue.

[0025] The impact of the combination therapy on TME reprogramming was assessed post- treatment. The combination therapy reversed the immunosuppressive TME, increasing CD4+ and cytotoxic CD8+ T cell infiltration by 6-fold and 11.4-fold, respectively, while collagen degradation. In addition, the combination therapy strategy also significantly reduced CD206+ macrophages and Foxp3 + Treg cell infiltration by 65.5% and 91%, respectively.

[0026] Therefore, the present application also provides the use of a substance having inhibitory effect on EZH2 / DNMT gene or its expression product in the preparation of a product for reversing the immunosuppression caused by castration-resistant prostate cancer.

[0027] As a preferred mode of the above technical solution, in the use of a substance having inhibitory effect on EZH2 / DNMT gene or its expression product in the preparation of a product for reversing the immunosuppression caused by castration-resistant prostate cancer, the product has the following effects:

[0028] (1) reversing the immunosuppressive tumor microenvironment, and / or

[0029] (2) increasing CD4+ cell infiltration, and / or

[0030] (3) increasing cytotoxic CD8+ T cell infiltration, and / or

[0031] (4) reducing CD206+ macrophage infiltration, and / or

[0032] (5) reducing Foxp3+ Treg cell infiltration, and / or

[0033] (6) enhancing the anti-tumor immune effect of existing immune checkpoint inhibitors (such as anti-PD-L1 / anti-PD-1).

[0034] Given the established role of EZH2 and DNMT in extracellular matrix remodeling, we evaluated collagen deposition by Masson's trichrome staining and picrosirius staining, and found that the combination therapy significantly reduced the collagen content shown by Masson's trichrome staining and picrosirius staining, respectively, indicating that it destroyed the tumor-promoting fibrotic microenvironment.

[0035] Therefore, the present application also provides the use of a substance having inhibitory effect on EZH2 / DNMT gene or its expression product in the preparation of a product for promoting collagen degradation of castration-resistant prostate cancer cells in vivo and / or inhibiting epithelial-mesenchymal transition of castration-resistant prostate cancer cells.

[0036] As a preferred embodiment of the above technical solution, the functional product / substance includes one or more of the following: EZH2 / DNMT nucleic acid inhibitor, EZH2 / DNMT protein inhibitor, immune-related cells with EZH2 / DNMT gene defects or silence, their differentiated cells, or gene recombinant constructs.

[0037] As a more preferred embodiment of the above technical solution, the functional product includes any one of the following:

[0038] (i) Small interfering RNAs, dsRNAs, shRNAs, microRNAs, and antisense nucleic acids that target EZH2 / DNMT or EZH2 / DNMT transcripts and can inhibit the expression of EZH2 / DNMT expression products or gene transcription;

[0039] (ii) It can express or form the small interfering RNA, dsRNA, shRNA, microRNA, and antisense nucleic acid constructs described in (i);

[0040] (iii) Contains an EZH2 / DNMT or EZH2 / DNMT complementary sequence, and is able to form an interfering molecule that inhibits the expression of EZH2 / DNMT gene expression products or gene transcription after being transfected into the body;

[0041] (iv) Immune-associated cells, their differentiated cells, or constructs after the EZH2 / DNMT gene sequence has been suppressed or knocked out.

[0042] Specifically, the functional products that inhibit the EZH2 gene or its expression product are GSK126, EZH2shRNA, and Tazemetostat (EPZ-6438), while the functional products that inhibit the DNMT gene or its expression product are DAC and Azacitidine.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] This invention reveals a dynamic epigenetic-extracellular matrix (ECM) co-evolutionary axis that drives the progression and treatment resistance of castration-resistant prostate cancer (CRPC). We demonstrate that dual targeting of DNMT and EZH2 disrupts this epigenetic shift, resensitizing the tumor to androgen deprivation therapy (ADT). Importantly, this strategy not only eliminated CRPC in preclinical models (tumor suppression rate of 90%, P<0.001) but also reversed immunosuppression, resulting in an 11.4-fold increase in cytotoxic T-cell infiltration. These findings redefine CRPC as a disease of chromatin-matrix interactions and establish a paradigm for joint targeting of epigenetic plasticity and the biomechanical microenvironment.

[0045] Our data expand the applicability of EZH2 inhibitors beyond PRC2-dependent cancers, pinpointing the interaction of DNA methyltransferases with EZH2 as a targetable vulnerability in extracellular matrix-rich solid tumors. This combination therapy offers a mechanistic solution to overcome stromal barriers—a transformative strategy for biomechanically driven malignancies. Attached Figure Description

[0046] Figure 1 High expression of EZH2 and DNMT in PCa is shown and associated with poorer clinical prognosis; (A) Comparison of EZH2, DNMT1, DNMT3A, and DNMT3B between PCa tumors (n=497) and normal tissues (n=52) in the TCGA database; data analysis was performed by UALCAN; (B) Kaplan-Meier survival plots of PCa individuals with high and low expression of EZH2, DNMT1, DNMT3A, and DNMT3B (data from TCGA); data were analyzed using log-rank test on the GEPIA2 platform, with the number of individuals and p-values ​​indicated in the images; (C) Data analysis on the GEPIA2 platform. The correlation between EZH2 and different DNMTs in PCa tumor tissue was analyzed (data from TCGA); (D) Representative images of immunohistochemical staining of 5mC and H3K27me3 in collected normal tissue and PCa samples, magnification shown in the figure; (E) Analysis of IHC scores of 5mC and H3K27me3 in the specimen of Figure D; (F) Representative images of immunohistochemical staining of 5mC and H3K27me3 in matched normal tissue and tissue microarray (TMA) PCa samples, magnification shown in the figure; (G) IHC score analysis of 5mC and H3K27me3 in the specimen of Figure F; (H) Correlation between IHC staining of 5mC and H3K27me3 and clinical outcomes, including Gleason score (I) and tumor size (J) in the specimen of Figure F;

[0047] Figure 2Epigenetic transitions between H3K27me3 and DNA methylation in CRPCs; (A) Immunoblot analysis of H3K27me3 levels in DU145, PC-3, and / or TRAMP-C1 cells treated with DMSO, GSK126 (10 μM), decitabine (1 μM), and a combination treatment (Combo); (B) Immunoblot analysis of EZH2 and H3K27me3 levels in DU145, PC-3, and TRAMP-C1 cells treated with DMSO and decitabine (1 μM) after EZH2 knockdown; (C) Calculated cytosine methylation rates of the CG, CHG, and CHH motifs, where H equals A, T, or C. Methylation rate = methylation reading / (methylation reading + unmethylation reading); (D) PCA analysis of gene methylation changes in the treatment group; (E) Violin plot of DMR distribution in the hypomethylation and hypermethylation groups; (F) Mean 5mC levels of different functional gene regions of the genome, including Up 2k, 5ʹ UTR, exons, introns, 3ʹ UTR, and Down 2k; (G) Immunoblotting analysis of EZH2, H3K27me3, and DNMT1 levels in DU145 cells after DNMT1 knockdown; (H) qPCR analysis of EZH2 expression in DU145 cells after DNMT1 knockdown; (I) Immunoblotting analysis of EZH2, H3K27me3, and DNMT1 levels in DU145 cells after EZH2 knockdown; (J) qPCR analysis of DNMT1 expression in DU145 cells after EZH2 knockdown;

[0048] Figure 3 The regulatory relationship between DNMT3A, DNMT3B and EZH2 is shown; (AB) Western blot (WB) showed that EZH2 did not change after knockdown of DNMT3A (A) and DNMT3B (B); (CD) EZH2 knockdown enhanced the expression levels of DNMT3A (C) and DNMT3B (D) as detected by qPCR.

[0049] Figure 4The study demonstrated the synergistic inhibitory effect of co-targeting EZH2 / DNMT on the proliferation and metastasis of castration-resistant prostate cancer (CRPC); (A) Western blot (WB) showed apoptosis induction in CRPC cells treated with GSK126 (EZH2 inhibitor, 5 μM), decitabine (DAC, DNMT inhibitor, 0.5 μM), or a combination thereof; (B) Cell viability assay (CCK-8). The combination treatment showed a significant inhibitory effect compared to GSK126 or DAC alone (****P<0.0001; mean ± SEM); (CD) Colony formation assays showed that the combination treatment almost completely eradicated the clonogenicity of CRPC cells (98% reduction compared to control; *P<0.001; n=3 independent experiments); (EF) In cells treated with the combination, Transwell migration and matrix gel invasion assays showed significant inhibition of motility and invasiveness, respectively (*P<0.05 vs. single drug; n=3); (G) Synergistic quantification was performed using the Chou-Talalay combination index (CI): CI<1 indicates a synergistic effect of multiple dose combinations; unless otherwise stated, data represent three independent experiments.

[0050] Figure 5 The dual-targeting synergistic effect of EZH2 and DNMT inhibits the occurrence of prostate tumors (a xenograft subcutaneous tumor model derived from prostate cancer cells) and remodels the tumor microenvironment; (A) Schematic diagram of the experiment: Prostate cancer (PCa) cell (DU145)-derived xenograft model in immunodeficient mice treated with stable EZH2 knockdown (shRNA) and decitabine (DAC, 0.5 mg / kg); (BD) In ​​the xenograft model, EZH2 knockdown alone moderately reduced tumor volume and weight (18% reduction compared to control), while DAC monotherapy achieved 60% inhibition; the combination therapy induced near-complete tumor regression (92% weight loss, mean ± SEM, n=5); (EF) Histopathological analysis (E) and immunofluorescence (IFC) staining (F) showed that the combination therapy increased apoptotic bodies and cleaved caspase-3+ cells in the tumor, while inhibiting Ki-67+ proliferating cells;

[0051] Figure 6 The experiment shows the safety of dual-targeted synergistic therapy of EZH2 and DNMT in mice; (A) Histological assessment of organ damage after treatment by HE staining showed no damage to organs such as heart, liver, spleen, lung, and kidney; (B) The change in body weight of mice after treatment was not significantly different from that of the control group, the drug toxicity was negligible, and the safety was high; Vehicle was the control group (using the solvent to dissolve DAC).

[0052] Figure 7The dual targeting synergistic effect of EZH2 and DNMT inhibits prostate tumor development (eZH2 knockdown TRAMP-C1 cell synergistic subcutaneous model) and remodels the tumor microenvironment; (A) Schematic diagram of the experiment: Synergistic subcutaneous model of TRAMP-C1 cells using EZH2 shRNA in immunocompetent mice; (BD) In ​​the synergistic model, the combination therapy reduced tumor volume and weight by 93% and 95%, respectively (mean ± SEM, n=5); (EF) H&E and immunofluorescence confirmed enhanced apoptosis, with 8.5-fold lysis of cleaved caspase-3+ cells (*P<0.05) and 35% inhibition of Ki-67+ cell proliferation (*P<0.05).

[0053] Figure 8 The dual targeting of EZH2 and DNMT synergistically inhibits prostate tumorigenesis and remodels the tumor microenvironment; (AB) Collagen deposition (Mason staining and Sirius red staining) was reduced after dual treatment in a xenograft subcutaneous tumor model derived from prostate cancer cells (20x magnification); (CD) Collagen deposition (Mason staining and Sirius red staining) was reduced after dual treatment in a homologous subcutaneous model of EZH2 knockdown TRAMP-C1 cells (20x magnification); (E) IHC staining of CD8+ and CD206+ in 11 pairs of human prostate cancer specimens (40x magnification); (FG) IHC and IFC showed that the combination therapy reversed immunosuppression, increased CD4+ (6-fold) and CD8+ T cell infiltration (11.4-fold) (*P<0.05) (F) and reduced CD206+ macrophages and Foxp3+ Tregs in the tumor microenvironment (G).

[0054] Figure 9 Displaying the synergistic reactivation of anti-tumor immunity by co-targeting EZH2 and DNMT; (A) Principal component analysis (PCA) of total mRNA-seq for different phenotypes; (B) Row z-score heatmap of significantly upregulated genes (Log2FC>2 and FDR<0.01) in four different phenotypes; (CF) GSEA analysis of the HALLMARK gene set associated with immune response in the combination therapy group using differentially expressed genes from all other groups; Top: Row z-score heatmap of core enriched genes in the HALLMARK gene set; Bottom GSEA plots for Combo and REST, reporting measured NES and FDR values; (GH) GSEA plots of the viral mimic / PRC2-targeted gene set for the Combo and REST gene sets, reporting measured NES and FDR values. Detailed Implementation

[0055] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific drawings and embodiments. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0056] Methods and Materials

[0057] Patient Samples: This study collected prostate cancer tissue from 11 patients who underwent radical prostatectomy and selected 11 normal prostate tissue samples as controls. All specimens were histopathologically confirmed by H&E staining and independently reviewed by two urogenital pathologists. The prostate cancer tissue microarray was provided by Shanghai Chipchao Biotechnology Co., Ltd. (product number: HProA060PG01). Sample collection followed the research protocol approved by the Medical Ethics Committee of the Fifth Affiliated Hospital of Sun Yat-sen University (ethics approval number: 2024-K186-1), and written informed consent was obtained in accordance with the Declaration of Helsinki.

[0058] Combinatorial shRNA reporter gene assay: HEK293T cells were treated with lentivirus-mediated knockdown of EZH2 and / or DNMT1 (for 72 hours), with the combined knockdown protocol being sh-EZH2 + sh-DNMT1 at an infection efficiency ratio of 1:1 (1000 viral particles per μL of viral vector). A non-targeted control (sh-Ctrl) was also included. The knockdown effect was confirmed by qRT-PCR.

[0059] Mouse experiments: All mouse husbandry and experimental procedures were strictly conducted in accordance with the policies of the Laboratory Animal Ethics Committee of the Fifth Affiliated Hospital of Sun Yat-sen University (Approval Nos.: 00379, 00455, 00478). Nude mice and C57BL / 6 mice were first castrated. After a two-week recovery period, DU145-shEZH2 / shCtrl or TRAMP-C1-shEZH2 / shCtrl cells were injected subcutaneously into nude mice and C57BL / 6 mice, respectively. One week later, the animals were randomly assigned to treatment and control groups. DAC was administered intraperitoneally at a dose of 0.5 mg / kg twice weekly. Tumor volume was determined by measuring changes in tumor size over time using the formula "Volume = Length × Width² × 0.52".

[0060] Histopathological and fibrosis analysis: H&E staining, Masson staining, and Sirius Red staining were performed by Wuhan Saiwei Biotechnology Co., Ltd. Stained tissue sections were scanned and analyzed using a Panoramic 250 Flash III scanner (3DHISTECH). H&E-stained sections were used to assess apoptosis by counting abnormal cells. Masson trichrome and Sirius Red stained images were quantitatively analyzed using ImageJ software. Paraffin-embedded sections (4 μm) fixed in formalin underwent heat-induced antigen retrieval in EDTA buffer (pH 9.0, 100°C, 20 min), followed by endogenous peroxidase blocking (3% H2O2, 15 min) and protein blocking (5% normal goat serum, 30 min). The major antibodies were incubated overnight at 4°C: anti-CD8 (Servicebio, GB12068, 1:500), anti-CD206 (Servicebio, GB115273, 1:500), anti-Foxp3 (Servicebio, GB112325, 1:500), anti-CleavedCaspase-3 (Servicebio, GB11532, 1:600), and anti-Ki67 (Servicebio, GB121141, 1:200). Detection was performed using ImmPRESS® HRP polymeric secondary antibodies (Servicebio, GB23303 / GB23301) with DAB chromogenic reagent, followed by hematoxylin counterstaining.

[0061] Hydroxyproline assay: Subcutaneous tumors were obtained, weighed, and dried, and then their hydroxyproline (HYP) content was determined. The specific steps are as follows: Each sample was hydrolyzed with 6N HCl at 120°C and dried; the resulting precipitate was reconstituted with the HYP detection buffer in the hydroxyproline (HYP) content detection kit (Acmec, AC10115); 200 μl of the sample solution diluted at a ratio of 1:4 was taken, and 150 μl of chloramine T solution was added and reacted for 20 minutes; then 150 μl of aldehyde-perchloric acid solution was added and reacted at 60°C for 15 minutes; finally, the absorbance value was measured at 560 nm using an Epoch microplate spectrophotometer (BioTek Instruments Limited); based on the theoretical value that collagen contains 13.7% hydroxyproline, the measured HYP content was converted into collagen mass, and the collagen content ratio is the ratio of collagen mass to tissue mass.

[0062] RNA sequencing and bioinformatics analysis: Total RNA was extracted from cells using TRIzol (Sigma), and cDNA samples were sequenced on the DNBseq-T7 platform (BGI Genomics, Shenzhen, China). The sequencing strategy was 150 bp paired ends, and the sequencing depth for each sample was 2 × 10⁻⁶. 7 Sequencing data were filtered using SOAPnuke, and cleanreads were aligned to a reference gene set using Bowtie2. Gene expression levels were calculated using RSEM (v1.3.1). Based on gene expression differences among different samples, heatmaps were generated using pheatmap (v1.0.12). Key differential expression analysis was performed using DESeq2 (v1.34.0), with a screening criterion of Q value ≤ 0.05. To further explore phenotypic changes, GO (http: / / www.geneontology.org / ) and KEGG (https: / / www.kegg.jp / ) pathway enrichment analysis was performed on annotated differentially expressed genes using Phyper based on hypergeometric tests. The significance levels of significantly enriched GO entries and KEGG pathways were corrected for Q value, with a strict threshold of Q value ≤ 0.05. Weighted co-expression network analysis (WGCNA) was performed using the R package 'WGCNA' to identify the gene expression modules most relevant to different phenotypes.

[0063] Whole-genome DNA methylation analysis: Prostate cancer cells were treated with epigenetic regulatory drugs for 72 hours. A whole-genome bisulfite sequencing (WGBS) library was constructed using the Single Strand Bisulfite-Seq Library Prep Kit (Acegen, AG0312) according to the manufacturer's instructions. In short, more than 0.5 μg of genomic DNA was mixed with 1‰ unmethylated lambda DNA, sonicated to an average fragment size of approximately 100-500 bp, and then converted to bisulfite. Subsequently, the heat-denatured single-stranded DNA was ligated with 3'-dA-tailed adapters. The 3'-tailed single-stranded DNA was then extended to double strands and ligated with 5-methylcytosine-modified adapters. After bisulfite conversion, approximately 10 cycles of PCR amplification were performed using Illumina 8-bp paired-end index primers. The constructed WGBS library was analyzed using an Agilent 2100 Bioanalyzer, and finally analyzed in Illumina. Sequencing was performed on the Nova platform using a 150×2 paired-end sequencing strategy; this whole-genome bisulfite sequencing (WGBS) was completed by Beijing Qingke Biotechnology (Tianjin) Co., Ltd.

[0064] Chromatin sequencing analysis of epigenetic drug response: Drug-treated cells were sent to Active Motif (Shanghai) Co., Ltd. for ChIP-Seq analysis. The company was responsible for chromatin preparation, ChIP reaction, library construction, and basic data analysis. In short, chromatin was separated by adding lysis buffer and sonicated. DNA was sheared to an average length of 200–500 bp using the EpiShear probe sonicator (catalog number 53051). Genomic DNA (Input) was decrosslinked at 65°C for 4 hours, digested with RNase A (Thermo Fisher) and Proteinase K (Thermo Fisher), purified using a QIAGEN PCR purification kit, and quantified using Onedrop (Wins). Fragmented chromatin for IP was incubated overnight at 4°C with 5 μL of anti-H3K27me3 antibody (Active Motif, catalog number 39155). 25 μL of Protein AMagarose Beads (smart-lifesciences) were added. Incubate at 4°C for 2 hours; after washing, the complex is eluted from the magnetic beads with SDS buffer and then decrosslinked overnight at 65°C; the decrosslinked chromatin is treated with RNase A and Proteinase K, and the ChIP DNA is purified using the QIAGEN PCR purification kit and quantified using Qubit (Thermo Fisher); Illumina sequencing libraries are constructed using ChIP-DNA and input DNA as templates using the VAHTSU Universal DNA Library Prep Kit for Illumina V4; after multiplexing, the libraries are sequenced on the Illumina NovaSeq 6000 S4 Reagent Kit V1.5 (300 cycles) on the Illumina NovaSeq 6000 platform; finally, visualization analysis is performed using the Integrative Genomics Viewer software (IGV, version 2.16.0).

[0065] Data Analysis: Data are presented as mean ± standard error. Statistical analysis was performed using GraphPad Prism 9.0; see the legend for details. A p-value < 0.05 was considered statistically significant. Statistical significance is indicated by asterisks as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0066] Example 1: Negative correlation of H3K27me3-DNA methylation can predict poor prognosis and epigenetic plasticity in prostate cancer.

[0067] We evaluated the prognostic relationship between a prostate cancer cohort and epigenetic modifications. First, we analyzed a previously reported Cancer Genome Atlas (TCGA) cohort of prostate cancer patients, observing significantly higher levels of EZH2, DNMT3A, and DNMT3B in 497 prostate cancer patients compared to 52 healthy individuals. Figure 1 A). Furthermore, even when statistical significance was not reached, a trend was observed where DNMT1 levels were higher in prostate cancer tissue than in normal tissue. Figure 1 A). Furthermore, high levels of EZH2 (p=3.8×10⁻⁶) were observed. -4 ), DNMT1 (p=3×10) -5 ), DNMT3A (p=3.9×10) -2 ) and DNMT3B (p=2.4×10 -3 The expression level of this substance is associated with shorter survival, indicating a poor clinical prognosis for prostate cancer. Figure 1 B). Notably, EZH2 expression was positively correlated with the expression of DNMT1, DNMT3A, and DNMT3, respectively. Figure 1 C).

[0068] Given the enzymatic activities of EZH2 and DNMTs, we hypothesized a positive correlation between H3K27me3 and DNA methylation in prostate cancer. However, this positive correlation with the presence of EZH2 and DNMTs at the mRNA level ( Figure 1 C) Conversely, immunohistochemical staining (IHC) results in clinical samples showed a negative correlation between H3K27me3 and DNA methylation. Figure 1 DE indicates a dynamic transition among these inhibitory markers during disease progression. To further rule out sample differences, we performed immunohistochemical staining on prostate cancer tissue microarrays and again observed a similar negative correlation between H3K27me3 and DNA methylation in PCa. Figure 1 FG). Furthermore, H3K27me3 levels showed a positive correlation with Gleason scores (FG). Figure 1 H), while 5mC (DNA methylation marker) level is related to Gleason score ( Figure 1 I) and tumor volume ( Figure 1 The negative correlation between J) further suggests a negative correlation between H3K27me3 and DNA methylation.

[0069] These combined data suggest that the negative correlation between H3K27me3 and DNA methylation is associated with poor clinical prognosis in prostate cancer.

[0070] Example 2: Bidirectional compensatory switching of H3K27me3 and DNA methylation drives CRPC

[0071] First, by detecting the level of endogenous H3K27me3 in prostate cancer cell lines, we investigated whether targeting DNMT enhances EZH2 activity. We observed an EZH2-dependent increase in H3K27me3 after decitabine (DAC, a DNMT inhibitor) treatment. Figure 2 A). To rule out off-target effects of EZH2 inhibitors, we knocked down endogenous EZH2 before DAC treatment. Similarly, DNMT inhibitors reversed EZH2-dependent H3K27me3 levels ( Figure 2 B).

[0072] To further elucidate the interaction between H3K27me3 regulation and DNA methylation (5mC), we performed whole-genome bisulfite sequencing (WGBS) on castration-resistant prostate cancer (CRPC) cells under different treatment conditions. The results showed that methylation mainly occurred on cytosine in CpG dinucleotides (CG), with very little cytosine methylation observed at CHG or CHH sites. Figure 2 C). Furthermore, GSK126 treatment altered the global DNA methylation pattern. GSK126 treatment alone increased genome-wide CpG methylation levels from 37% to 39% (C). Figure 2 C). Furthermore, prior treatment with the DNMT inhibitor DAC followed by GSK126 treatment resulted in a more significant increase in CpG methylation, with the methylation level rising from 18% to 25%. Figure 2 C). Principal component analysis (PCA) further confirmed significant differences in CpG methylation profiles among different treatment groups. Figure 2 D).

[0073] Differential methylation region (DMR) analysis comparing the GSK126-treated group and the DMSO control group revealed that 61.24% of the DMRs (262,523 regions) were hypermethylated (hypermethylated DMRs), and 38.76% (166,135 regions) were hypomethylated (hypomethylated DMRs). Figure 2 E). This indicates that GSK126 treatment primarily drives genome-wide hypermethylation. Furthermore, GSK126 treatment increased 5mC levels in all genomic regions (DMSO group vs. GSK126 group), including the 2 kb region upstream of transcription start sites (Up 2k), 5' untranslated regions (5' UTRs), exons, introns, 3' untranslated regions (3' UTRs), and the 2 kb region downstream of transcription termination sites (Down 2k). Figure 2F). In cells pretreated with DAC, subsequent GSK126 treatment resulted in a more significant increase in 5mC levels (Combo group vs. GSK126 group). Figure 2 F).

[0074] We found that inhibiting EZH2 significantly enhanced global DNA methylation levels, especially after DNMT inhibition. These results suggest that DNMT inhibitors promote H3K27me3 accumulation in an EZH2-dependent manner; conversely, inhibiting EZH2 increases genome-wide DNA methylation of CpG islands, revealing a bidirectional compensatory mechanism.

[0075] Given the crucial role of DNMTs in DNA methylation, the specific DNMT subtypes mediating the above phenomenon were then identified. shRNA-mediated gene knockdown experiments determined that DNMT1 (but not DNMT3A / B) was the main regulator of H3K27me3 increase. Figure 2 GH and Figure 3 AB), thus establishing the DNMT1-EZH2 interaction as a core axis of epigenetic plasticity. Furthermore, knockdown of EZH2 increased the levels of DNMT1, DNMT3A, and DNMT3B (AB). Figure 2 IJ and Figures 3C-D).

[0076] These combined data indicate that in CRPC, there is a bidirectional compensatory switching between H3K27me3, regulated by EZH2 and DNMT1, and DNA methylation.

[0077] Example 3: Dual targeting of EZH2 and DNMT induces synergistic antitumor activity in multiple CRPC models

[0078] Given the observed bidirectional compensatory switching between H3K27me3 and DNA methylation, we hypothesize that simultaneous targeting of EZH2 and DNMT is a novel therapeutic strategy for synergistically inhibiting CRPC.

[0079] To test this hypothesis, we evaluated the effect of combined GSK126 and DAC on castration-resistant prostate cancer cells. Compared with monotherapy, the combination of GSK126 and DAC (combination treatment) significantly increased cell apoptosis. Figure 4 A). Consistent with this, the combined treatment significantly inhibited cell viability ( Figure 4 B), and the number of cell colonies formed is even smaller ( Figure 4 CD). Furthermore, compared to the monotherapy group, the combination of GSK126 and DAC (combination treatment) significantly reduced cell migration levels, indicating weakened cell motility after treatment. Figure 4 E). Furthermore, the combined treatment significantly inhibited cell invasion ability (Figure 4 F). Near-complete inhibition of invasion and migration indicates a disruption of metastatic capacity, a key characteristic of treatment-resistant CRPC. To quantify the synergistic effect of these combinations, we calculated the combination index (CI) of GSK126 with DAC. The combination index (CI) of GSK126 with decitabine ranged from 0.6 to 0.8. Figure 4 G), and synergistic effects of the drugs were demonstrated in multiple dosage regimens and cell lines (CI<1).

[0080] Example 4: Dual epigenetic targeting inhibition of EZH2 and DNMT can synergistically inhibit prostate cancer progression and regulate anti-tumor immunity in vivo.

[0081] To evaluate the therapeutic potential of dual epigenetic targeting, we used a subcutaneous xenograft model of prostate cancer cells (the modeling process is as follows). Figure 5 A). To mitigate the potential off-target effects of the pharmacological EZH2 inhibitor (GSK126), we employed a stable genetic approach (sh-EZH2) to knock down EZH2. Knockdown of EZH2 alone only moderately reduced tumor volume ( Figure 5 However, when combined with the DNMT inhibitor decitabine (DAC, 0.5 mg / kg), it produced a synergistic effect in inhibiting tumor growth: the tumor volume in the combination therapy group was reduced by 90% compared with the control group (P<0.001). Similarly, the tumor weight in the combination therapy group was also significantly lower than that in the monotherapy group (sh-EZH2: reduction of 18%; DAC: reduction of 60%; combination therapy: reduction of 92%; P<0.001). Figure 5 D).

[0082] It is noteworthy that the combination therapy regimen did not exhibit drug toxicity, which was confirmed by the absence of significant changes in body weight and normal organ and tissue sections. Figure 6 AB).

[0083] Histopathological analysis showed that the tumors treated with the combination therapy exhibited significant apoptotic features, including cytoplasmic condensation and nuclear pyknosis, and the number of apoptotic bodies increased by 3.4 times and 1.6 times, respectively, compared with the EZH2 knockdown monotherapy group and the DAC monotherapy group (P<0.001). Figure 5 E). Immunofluorescence results confirmed these findings: the number of cleaved caspase-3 positive cells surged 16.7-fold in the combination therapy group (P<0.001), while Ki67 positive proliferating cells decreased by 91% (P<0.001). Figure 5F). Given the established roles of EZH2 and DNMT in extracellular matrix remodeling, we assessed collagen deposition using Masson and Sirius red staining, and found that the combination therapy significantly reduced collagen content as indicated by both Masson and Sirius red staining. Figure 8 AB) indicates that it disrupts the tumor-promoting fibrotic microenvironment.

[0084] To investigate the immunomodulatory effects, we established a subcutaneous model of TRAMP-C1 cells derived from sh-EZH2 in immunocompetent mice (modeling process). Figure 7 A). Although DAC monotherapy or EZH2 knockdown alone showed only modest efficacy, their combination reduced tumor volume and weight by 93% and 95%, respectively (P<0.001, Figure 7 BD). H&E and immunofluorescence analysis results were consistent with previous findings, showing an 8.5-fold increase in apoptosis and a 35% inhibition of proliferation. Figure 7 EF). Collagen degradation is further aggravated in this model ( Figure 8 CD). Given the presence of an immunosuppressive tumor microenvironment (TME) in tumors, characterized by a decrease in cytotoxic CD8⁺ T cells and an increase in CD206⁺ macrophages compared to normal tissues (CD). Figure 8 E).

[0085] We evaluated the effect of combination therapy on TME reprogramming. Combination therapy reversed immunosuppressive TME, increasing the infiltration of CD4⁺ and cytotoxic CD8⁺ T cells by 6-fold and 11.4-fold, respectively, while simultaneously degrading collagen (P<0.01). Figure 8 F). Furthermore, this combination therapy strategy also significantly reduced CD206⁺ macrophages and Foxp3. + Treg cell infiltration was reduced by 65.5% and 91%, respectively (P<0.01 compared with the control group). Figure 8 G).

[0086] In summary, these data suggest that dual epigenetic targeting of EZH2 and DNMT can disrupt pro-tumor fibrotic niches and reverse immunosuppression.

[0087] Example 5: Dual Inhibition of EZH2 / DNMT Reversal of Immunosuppression

[0088] Given the immunomodulatory effects of the combined treatment observed in animals (Example 4), we investigated its potential transcriptional mechanisms. RNA sequencing (RNA-seq) revealed unique transcriptional profiles among the different treatment groups. Figure 9A). Although monotherapy with DAC or GSK126 upregulated the expression of 443 and 71 response genes, respectively, combination therapy upregulated the expression of 631 response genes. Figure 9 B). Gene set enrichment analysis (GSEA) of the combination therapy group and the control group (vehicle / GSK126 / DAC) showed significant enrichment of immune activation pathways, including inflammatory responses ( Figure 9 C), IL-6 / JAK / STAT3 signaling pathway ( Figure 9 D), Interferon α ( Figure 9 E) and interferon-γ ( Figure 9 F).

[0089] Combination therapy upregulated viral mimicry-related innate immune pathways. Figure 9 G) and PRC2 target genes ( Figure 9 Transcriptome analysis revealed synergistic activation of interferon responses and PRC2 target genes, explaining persistent immune reactivation. In summary, these data demonstrate that dual inhibition of EZH2 / DNMT reverses tumor immunosuppression by activating a therapeutically viable innate immune pathway.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. The use of Product A in the preparation of kits or reagent kits for improving the accuracy of clinical diagnosis or prognosis in patients with castration-resistant prostate cancer, characterized in that, Product A includes substances for detecting the expression levels of the EZH2 gene and / or the DNMT gene.

2. The application of the EZH2 / DNMT gene or its expression product in the development and screening of functional products for castration-resistant prostate cancer, characterized in that, The functional product has an inhibitory effect on the EZH / DNMT gene or its expression product; the functional product can break the castration-resistant prostate cancer driven by DNA methylation and compensatory epigenetic transformation of H3K27me3.

3. Application of functional products that inhibit the EZH2 / DNMT gene or its expression products in the preparation of products for the treatment of castration-resistant prostate cancer.

4. The application according to claim 2 or 3, characterized in that, When the functional product is used externally, the functional product has the following functions: (1) Promotes apoptosis in castration-resistant prostate cancer cells, and / or (2) Inhibits the proliferation of castration-resistant prostate cancer cells, and / or (3) Inhibits the migration of castration-resistant prostate cancer cells, and / or (4) Inhibits the invasion of castration-resistant prostate cancer cells; When the functional product is used in the body, the functional product has the following effects: (1) Inhibit tumor growth, reduce tumor volume and / or (2) Degrade the extracellular matrix of tumor tissue and reduce tumor stiffness.

5. Application of substances that inhibit the EZH2 / DNMT gene or its expression product in the preparation of products that enhance the antitumor immune effects of existing immune checkpoint inhibitors and thereby reverse immunosuppression caused by castration-resistant prostate cancer.

6. The application according to claim 5, characterized in that, The product has the following functions: (1) Reversing the immunosuppressive tumor microenvironment, and / or (2) Increase CD4+ cell infiltration, and / or (3) Enhances cytotoxic CD8+ T cell infiltration, and / or (4) Reduce CD206+ macrophage infiltration, and / or (5) Reduce Foxp3+ Treg cell infiltration, and / or (6) Enhance the anti-tumor immune effect of existing immune checkpoint inhibitors.

7. Application of substances that inhibit the EZH2 / DNMT gene or its expression product in the preparation of products that promote collagen breakdown in castration-resistant prostate cancer cells and / or inhibit epithelial-mesenchymal transition in castration-resistant prostate cancer cells.

8. The application according to claim 4, or claim 5, or claim 6, or claim 7, characterized in that, The functional products / substances include one or more of the following: EZH2 / DNMT nucleic acid inhibitors, EZH2 / DNMT protein inhibitors, immune-related cells with EZH2 / DNMT gene defects or silence, their differentiated cells, or gene recombinant constructs.

9. The application according to claim 8, characterized in that, The functional product includes any of the following: (i) Small interfering RNAs, dsRNAs, shRNAs, microRNAs, and antisense nucleic acids that target EZH2 / DNMT or EZH2 / DNMT transcripts and can inhibit the expression of EZH2 / DNMT expression products or gene transcription; (ii) It can express or form the small interfering RNA, dsRNA, shRNA, microRNA, and antisense nucleic acid constructs described in (i); (iii) Contains an EZH2 / DNMT or EZH2 / DNMT complementary sequence, and is able to form an interfering molecule that inhibits the expression of EZH2 / DNMT gene expression products or gene transcription after being transfected into the body; (iv) Immune-associated cells, their differentiated cells, or constructs after the EZH2 / DNMT gene sequence has been suppressed or knocked out.

10. The application according to claim 9, characterized in that, Functional products that inhibit the EZH2 gene or its expression product include GSK126, shRNA-EZH2, and Tazemetostat (EPZ-6438), while functional products that inhibit the DNMT gene or its expression product include DAC and Azacitidine.

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