Application of PD-1 inhibitor in preparation of medicine for treating prostatic cancer with TP53 gene mutation

Abstract

The invention provides application of a PD-1 inhibitor to preparation of a medicine for treating prostatic cancer with TP53 gene mutation, and relates to the technical field of medicine. The medicine comprises a therapeutically effective amount of the PD-1 inhibitor and a pharmaceutically acceptable carrier auxiliary component thereof. The PD-1 inhibitor and Mutp53 are related to epithelial lineage, metabolic reprogramming and recombination of CAF and immune cell population, so that the improvement of tumor immunity accessibility is promoted together. In a comprehensive view, the discovery reveals a direct mechanism relation among TP53 high-frequency mutation, Cxcl10 signal transduction and ICB treatment sensitivity, and a new biological insight and a potential strategy are provided for layering and combined treatment of patients suffering from CRPC.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of medicines, in particular to application of a PD-1 inhibitor in preparation of a medicine for treating prostate cancer with TP53 gene mutation. BACKGROUND

[0002] Prostate cancer is a major global health problem and one of the leading malignancies affecting men worldwide. TP53 gene is frequently mutated in prostate cancer and many other tumor types, which has been proven to be related to genomic integrity, programmed cell death and cell cycle regulation for a long time, and such an immunosuppressive microenvironment is generally considered to reduce the reactivity to immunotherapy.

[0003] Contradictorily, a plurality of clinical studies show that patients carrying TP53 mutation are more favorable to PD-1 blockage. Such a difference between "biological expectation" and "clinical observation" indicates that TP53 mutation has a context-dependent effect, which drives an immunosuppressive program on one hand and simultaneously activates immune pathways, so that tumors are sensitive to checkpoint blockage under certain conditions. Supporting this point of view is that, in non-small cell lung cancer, KRAS and TP53 co-mutation is closely related to increased PD-1 expression, enhanced CD8+ T cell infiltration and improved effect of PD-1 inhibitor treatment. SUMMARY

[0004] In view of the deficiencies in the prior art, the application provides application of a PD-1 inhibitor in preparation of a medicine for treating prostate cancer with TP53 gene mutation, in particular to the following: Application of a PD-1 inhibitor in preparation of a medicine for treating prostate cancer with TP53 gene mutation.

[0005] Preferably, the medicine comprises a therapeutically effective amount of the PD-1 inhibitor and a pharmaceutically acceptable carrier auxiliary component.

[0006] Preferably, the TP53 gene mutation is any one of missense mutation, gene deletion, frameshift mutation, splice site mutation and truncation mutation.

[0007] Preferably, the inhibitor is any one of a PD-1 inhibitor, a Glafenine inhibitor, a LY3522348 inhibitor and an AMG487 inhibitor.

[0008] The application provides application of a PD-1 inhibitor in preparation of a medicine for treating prostate cancer with TP53 gene mutation, which has the following advantages compared with the prior art: Mutp53 is associated with epithelial lineage, metabolic reprogramming, and reorganization of CAF and immune cell populations by PD-1 inhibitors, collectively contributing to enhanced tumor immune accessibility. Taken together, these findings reveal a direct mechanistic link between TP53 hypermutation, Cxcl10 signaling, and ICB treatment sensitivity, providing new biological insights and potential strategies for patient stratification and combination therapy in CRPC. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 A schematic diagram of CRISPR / Cas9-mediated knock-in of Trp53 p.R245Q (arginine to glutamine) in prostate cancer cells, as well as the workflow of in vivo validation and single-cell RNA sequencing; B-D are the subcutaneous tumor growth of Myc-CaP cells in FVB / NJ mice; B is a representative image of tumors at the endpoint; C is the longitudinal tumor growth curve during the study; D is the final tumor weight after sacrifice; E-G are the subcutaneous tumor growth of RM-1 cells in C57BL / 6 mice, where E is a representative image of tumors at the endpoint; E is the longitudinal tumor volume curve; E is the final tumor weight after sacrifice; H is a schematic diagram of the Kaplan-Meier survival duration of subcutaneously tumor-bearing mice; I is a point plot visualization of the expression of classical marker genes in major cell types (including epithelial cells, endothelial cells, monocyte / macrophages, T / NK cells, fibroblasts, and pericytes) in the single-cell transcriptome data set; J is a UMAP visualization of the single-cell transcriptome; K is the relative abundance of major cell populations in the tumor microenvironment; L is a heatmap of the observed-to-expected ratio of major cell populations; M is a multiplex immunofluorescence staining of tumor sections, labeling Pan-CK, CD4, FoxP3, CD8, CD68, CD163, and CTLA-4; Figure 2Figure 6. Mutp53 epithelial cells are enriched in genes associated with arachidonic acid and fructose / mannose metabolism. A, UMAP plot showing reclassification of epithelial cells from WTp53 and Mutp53 tumors into seven distinct transcriptional states (C0-C6); B, Box plot showing the relative abundance of each epithelial cluster in different genotypes; C, Heatmap showing the dominant genes of each cluster, with associated Gene Ontology (GO, left) and Kyoto Encyclopedia of Genes and Genomes (KEGG, right) pathway enrichment results; D, Schematic of pseudotime inference results from Monocle3 trajectory analysis; E, Violin plot showing significant increase in arachidonic acid and fructose / mannose metabolism scores in Mutp53 epithelial cells (both p < 2 x 10-16, Wilcoxon rank-sum test); F, Schematic of CCK-8 proliferation assay results; G, Representative images of WTp53 and Mutp53 cells stained with EdU after 48 hours of Glafenine inhibitor treatment, scale bar, 50 pm; H, Quantitative analysis showing significant reduction in Mutp53 cell proliferation compared to control cells after LY3522348 inhibitor treatment (one-way ANOVA with Tukey’s post-hoc pairwise comparison) Figure 3 Figure 7. Anti-PD-1 treatment reduces Mutp53 tumor growth. A, Schematic of experimental design for subcutaneous and orthotopic RM-1 tumor models treated with anti-PD-1 antibody; B-D, Differences in WTp53 and Mutp53 subcutaneous tumors with and without anti-PD-1 treatment (two-way ANOVA with Tukey’s multiple comparison test) schematics, B, Representative images; C, Tumor volume curves; D, End-point tumor weights; E-G, Differences in orthotopic tumor growth after PD-1 blockade treatment schematics, E, Representative images; F, Tumor volumes; G, End-point tumor weights; H, Flow cytometry analysis of CD8+GZMB+T cells within tumors at baseline and after PD-1 blockade; I, Quantitative results shown (one-way ANOVA with Tukey’s multiple comparison test); J-K, Single-cell RNA sequencing clustering of CD8+T cell subsets; L-S, Schematic of functional module score analysis of CD8+T cells, L-M, Co-stimulatory signaling results between groups; N-O, Exhaustion characteristics, R-S, Residency characteristics comparable, P-Q, Cytotoxicity characteristics in Mutp53 tumors schematics; T, Expression of representative effector genes in CD8+T cells within tumors; U, Multiple immunofluorescence staining of CD8 and GZMB showing; Figure 4Fig. 9. Cxcl10 is a p53 target gene and is upregulated in Mutp53 tumors. A-B, Heatmap and qRT-PCR analysis of chemokine expression in WTp53 and Mutp53 tumors in subcutis. Cxcl10, Cxcl1, Cxcl2, Cxcl12, Ccl2 and Ccl9 were significantly upregulated in Mutp53 tumors compared with WTp53 tumors (n=5 for each group); C, Correlation analysis of Cxcl10 expression and immune-related pathways; D, Representative IHC staining results of Cxcl10 in WTp53 and Mutp53 tumors; E, Western blotting of Cxcl10 protein expression in Mutp53 and WTp53 cell lines and tumor tissues; F, Quantitative analysis; G, Transwell co-culture experiment showed that CD8+ T cells had stronger chemotactic migration ability to Mutp53-derived tumor cells than to WTp53-derived tumor cells (n=3); H, Clinical correlation of Cxcl10 expression level with ICB efficacy in IMvigor210 cohort. High expression of Cxcl10 can significantly predict better immunotherapy response; I, Schematic diagram of in vivo experiment design for pharmacological inhibition of Cxcl10-CXCR3 axis using AMG-487 during PD-1 blockade treatment; J-L, Schematic diagram of WTp53 and Mutp53 tumor-bearing mice after receiving anti-PD-1 treatment or combination with AMG-487 treatment, J, Representative tumor images, K, Tumor growth curve; L, Tumor weight at endpoint (n=5-7 for each group); Figure 5 Fig. 10. Cxcl10 is a p53 target gene and is upregulated in Mutp53 tumors. A, Western blotting results of RM-1 tumors showed that siRNA-mediated Trp53 silencing can reduce Cxcl10 protein levels, p53 and GAPDH were used as target protein control and loading control, respectively; B, JASPAR motif analysis identified potential p53 binding sequences within the Cxcl10 promoter. The positions of primer 1 and primer 2 relative to the transcription start site (TSS) and exon 1 (Exon 1) are shown; C-D, ChIP-qPCR analysis of p53 binding on the Cxcl10 promoter showed that C, Mutp53 enrichment in the primer 1 detection region; D, Mutp53 enrichment in the primer 2 detection region; E, Mutp53 enrichment in the primer 1 detection region; F, Mutp53 enrichment in the primer 1 detection region; IgG was used as a negative control. Mutp53 tumors had increased enrichment of H3K4me3, while H3K27me3 and H3K36me3 were decreased, consistent with a permissive promoter environment; G, Agarose gel electrophoresis results of representative ChIP amplification products; Left, base pair (bp) molecular weight marker; H, Working model; Figure 6A is a waterfall plot of TLS-enriched versus TLS-depleted tumor mutational profiles in the TCGA-PRAD cohort; B, D, F are representative images (DAPI nuclear counterstain in blue) showing increased CD3+ (T cells, magenta), CD20+ (B cells, red), and CD31+ (endothelial cells, green) cells in Mutp53 tumors compared to WTp53 tumors; C, E, G are cell density distribution plots (cells per field) showing intergroup comparisons and significance results after correction for multiple testing; Figure 7 A is scRNA-seq-based UMAP clustering analysis; B is quantification of fibroblast subtypes in WTp53 and Mutp53 tumors; C is expression projection of marker genes (e.g. Acta2, Lox, Col15a1, S100a10, Cxcl12, etc.) in UMAP space; D-E are pseudotemporal trajectory analysis showing differentiation paths of fibroblasts; F is a pseudotemporal heatmap of fibroblast lineage progression showing dynamic expression patterns of extracellular matrix-related genes (e.g. Tnc, Sparcll, and Col12a1) and immune-related genes across different subtypes; G is multiplex immunofluorescence staining results of a-SMA (CAF marker, yellow), CD8 (red), and CXCL12 (magenta) in WTp53 and Mutp53 tumors; Figure 8 Model of Mutp53-driven immune reprogramming; Figure 9 A is a schematic of gene editing strategy; B is restriction enzyme digestion after polymerase chain reaction (PCR) followed by agarose gel electrophoresis analysis, left: clone 3D3; right: clone 1B5; each panel includes DNA size marker (ladder), wild type control (WT), and no template control (NTC, labeled as H20); C is Sanger sequencing validation; top sequencing plot shows sequence of clone 3D3, proving it is homozygous CGC→CAG mutation, bottom sequencing plot shows clone 1B5, which retains wild type sequence (CGC), confirming it is homozygous wild type genotype; target codon is highlighted in yellow; Figure 10A UMAP visualization of myeloid cell populations from WTp53 and Mutp53 tumors showing distinct clusters of monocytes and macrophages, including tumor-associated macrophages (TAMs); B a proportion analysis of myeloid subpopulations in individual tumor samples (T1-T3: Mutp53; W1-W3: WTp53); C expression of monocyte-specific markers (e.g. S100a8, S100a9) confirming the identity of the clusters; D expression of macrophage-associated markers (e.g. Apoe, C1qa, C1qb); E expression of TAM-associated markers (Cd86, Arg1, Cd163, and Il6); F gene ontology (GO) enrichment analysis for each myeloid subpopulation, bubble plot representing the normalized enrichment score (NES), gene set size, and statistical significance; G transcription factor (TF) activity analysis in myeloid subpopulations; H differential transcription factor activity between WTp53 and Mutp53 tumors; Figure 11 Single-cell metabolic pathway analysis of epithelial cells in mutant and wild-type TP53 tumors; Figure 12 A violin plot showing IFN-gamma (interferon-gamma) expression in CD8+ T cells from wild-type p53 (WTp53) and mutant p53 (Mutp53) tumors; B flow cytometry analysis of tumor infiltrating lymphocytes; C immunofluorescence staining of tumor sections, staining markers are DAPI (nuclei, blue), CD8 (green), and IFN-gamma (magenta), merged images showing fewer CD8+IFN-gamma+ cells in Mutp53 tumors, scale bar, 100 pm. DETAILED DESCRIPTION

[0010] Mouse prostate cancer cell lines RM-1 (CRL-3310™) and MyC-CaP (CRL-3255™) were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China); the cell lines provided by the bank were identified, and these cell lines were originally derived from the American Type Culture Collection (ATCC; MANASSAS, VA, USA). RM-1 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium, and MyC-CaP cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM); both media were supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific) and 1% penicillin-streptomycin (Procell, Wuhan, Hubei, China), and the cells were cultured in a humidified incubator at 37°C with 5% CO2.

[0011] Explanation of abbreviations: CAF, cancer-associated fibroblast; UMAP, uniform manifold approximation and projection; myCAF, myofibroblastic CAF; iCAF, inflammatory CAF; scRNA-seq, single-cell RNA sequencing; ECM, extracellular matrix; a-SMA, a-smooth muscle actin; WT p53, wild-type p53; Mut p53, mutant p53; DAPI, nuclear counterstain.

[0012] Example 1: 1. Related experiments: 1.1 Cell culture and reagents All cell lines were identified using short tandem repeat (STR) analysis and routinely tested to ensure the absence of mycoplasma contamination. At the time of experiment, cells were harvested using 0.25% trypsin-ethylenediaminetetraacetic acid when they reached 80-90% confluency and seeded at appropriate density. Detailed information of all antibodies, assay kits and reagents, including product name and manufacturer, is listed in Table S1: Table S1

[0013] 1.2 CRISPR-Cas9 editing of Trp53 gene The Trp53 gene in MyC-CaP cells was edited by CRISPR-Cas9 ribonucleoprotein (RNP) complex with single-stranded DNA (ssDNA) donor template (Genewiz). After assembling the guide RNA targeting exon 7 (5’-TGATGATGGTAAGGATAGGT-CGG-3’) with hSpCas9 protein, transfection was achieved by electroporation using Neon™ transfection system. Single-cell cloning was performed in 96-well plates 72 hours after transfection. Genomic DNA was extracted using Quick-DNA MicroExtraction Kit (Zymo Research, Irvine). PCR amplification was performed using Taq DNA polymerase (2 U), dNTPs (100 mM) and primers (250 nM each) flanking exon 7, as shown in Table S2: Table S2

[0014] PCR products were verified by agarose gel electrophoresis and sequenced on an ABI 3730xl DNA analyzer. The final cell lines contain the following genotypes: (+ / +), , , and Clones carrying the bi-allelic Trp53 mutation were expanded and used for subsequent experiments. We also used the Mutp53 RM-1 cell line that we previously described.

[0015] 1.3 CCK-8 assay Cell Counting Kit-8 was used to measure cell viability, following the manufacturer’s instructions. Briefly, cells (2*10³ per well) were seeded in 96-well plates in 100 μL of complete medium per well and left to adhere overnight. After the completion of each treatment, 10 μL of CCK-8 reagent was added to each well and incubated for 1 hour at 37°C, 5% CO2. Subsequently, the absorbance was measured at 450 nm using a microplate reader. Wells containing medium only (without cells) were used as blank controls. Each condition was set up in triplicate and cell viability was expressed as a percentage relative to the control group.

[0016] 1.4 5-ethynyl-2’-deoxyuridine (EdU) cell proliferation assay For each genotype, cells were divided into three treatment groups: DMSO (solvent control), LY3522348 (MedChemExpress, Shanghai, China) and Glafenine (MedChemExpress, Shanghai, China). Cells were cultured under the same conditions and EdU reagent (Beyotime, Shanghai, China) was added according to the manufacturer’s instructions for incubation. After the completion of the labeling, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 and subjected to EdU (Beyotime, C0071S) fluorescent staining using the appropriate detection kit. Nuclei were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI). Fluorescent images were acquired by fluorescence microscopy and the number of EdU-positive proliferating cells was determined for statistical analysis.

[0017] 1.5 Small interfering RNA (siRNA) transfection Trp53-specific siRNA was transfected into RM-1 cells using Lipofectamine™3000 transfection reagent (Invitrogen, L3000075) following the manufacturer’s instructions. Cells were collected 48 hours after transfection and lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors, followed by Western blot.

[0018] 1.6 Western blot Cell or tissue samples were lysed with RIPA lysis buffer containing protease inhibitors on ice. Protein concentration was determined using Bicinchoninic Acid (BCA) Protein Assay Kit (Thermo Fisher Scientific). Equal amount of protein was mixed with sodium dodecyl sulfate (SDS) loading buffer (Biouniquer) and boiled, and then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The PVDF membrane was blocked with QuickBlock™ Western Blocking Buffer (Biouniquer) for 20 minutes at room temperature.

[0019] The membrane was incubated with specific primary antibody overnight at 4°C, followed by incubation with horseradish peroxidase (HRP)-labeled secondary antibody (Cell Signaling Technology, Danvers, MA, USA) for 1 hour at room temperature. Protein bands were developed using chemiluminescence (ECL) reagent (Thermo Fisher Scientific) and ChemiDoc™ Imaging System, and analyzed for density using ImageJ software.

[0020] 1.7 Chromatin immunoprecipitation-real-time quantitative polymerase chain reaction (ChIP-qPCR) ChIP experiments were performed using BeyoChIP™ ChIP Kit (Protein A / G) with slight modifications according to the manufacturer’s instructions. Briefly, cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature, and quenched with 0.125 mol / L glycine for 5 minutes. After purification of nuclei, chromatin was digested by enzymatic method, followed by sonication to obtain DNA fragments of about 200-500 bp. Chromatin lysate was incubated with 5 μg of specific antibody (anti-p53 antibody, anti-H3K4me3 antibody, anti-H3K9me3 antibody, anti-H3K36me3 antibody, anti-H3K27me3 antibody, or immunoglobulin extracted from normal, non-immune rabbit serum) pre-bound to Protein A / G magnetic beads overnight at 4°C. Immunoprecipitated chromatin was washed, eluted, and then reverse-crosslinked at 65°C overnight, followed by digestion with proteinase K. DNA was purified by spin column, and qPCR analysis was performed using SYBR Green Master Mix (Takara). Primers used for qPCR were targeted to the promoter region of Cxcl10 gene. Data were normalized with 2% input DNA as internal control, and results were presented as fold enrichment relative to IgG control.

[0021] 1.8 Animal experiments Six-week-old male C57BL / 6 mice (Jiejia Yakang Biotechnology Co., Ltd., Nanjing, Jiangsu, China) were housed in a specific pathogen-free (SPF) environment, and experimental procedures were in accordance with institutional guidelines and ARRIVE recommendations. There were two ways to establish the RM-1 prostate cancer model: one was to mix 1 x 10 6 cells with Matrigel / phosphate-buffered saline (PBS) at a ratio of 1:1 (Shanghai Novus Biological Technology Co., Ltd.), and then subcutaneously injected into the right flank of mice; the other was to inject cells into the prostate of mice in situ.

[0022] When the tumor volume reached about 100 mm 3 , the mice were randomly divided into treatment and control groups. Treatment regimens included anti-PD-1 antibody (10 mg / kg, intraperitoneal injection; BioXcell, Lebanon, NH) and AMG487 (5 mg / kg, intraperitoneal injection; Meilun Biotech Co., Ltd.). Tumor monitoring and drug administration were performed by blinded technicians. All experimental procedures were approved by the Animal Ethics Committee of Anhui Medical University (Approval No. LLSC20241412).

[0023] 1.9 Hematoxylin-eosin (HE) staining and immunohistochemical staining Paraffin-embedded tissue sections were deparaffinated with xylene and then gradually rehydrated through graded ethanol solutions. For histological staining, cell nuclei were stained with hematoxylin, and then the cytoplasm was stained with eosin, followed by dehydration, transparency treatment, and mounting.

[0024] For immunohistochemical staining, antigen retrieval was performed in citrate buffer under high temperature and pressure conditions. The sections were pretreated with normal serum for 30 minutes, and then incubated with the specified primary antibody at 4°C overnight. The next day, the sections were incubated with HRP-labeled secondary antibody (Aibisheng Biotechnology Co., Ltd., Changzhou, Jiangsu, China) at room temperature for 1 hour, and then developed with the corresponding 3,3'-diaminobenzidine (DAB) substrate (Agilent Technologies). The nuclei were counterstained with hematoxylin, and then the sections were dehydrated, mounted, and finally observed under a light microscope.

[0025] 1.10 Multiplex immunofluorescence staining The samples were fixed with formaldehyde and then blocked with bovine serum albumin (Sigma-Aldrich) to avoid non-specific binding. The primary antibodies against Pan-CK, CD4, FoxP3, CD8, CD68, CD163, CTLA-4, and GZMB were applied to the tissue sections and incubated at 4°C overnight. Then, the sections were washed and incubated with fluorescently labeled secondary antibodies at room temperature. After the nuclei were counterstained with DAPI, the fluorescent signals were acquired by fluorescence microscopy.

[0026] The expression levels of CD3, CD20 and CD31 were quantitatively analyzed by immunofluorescence images. All images were exported in RGB format, and data processing was performed using Python software.

[0027] 1.11 Enzyme-linked immunosorbent assay (ELISA) Enzyme-linked immunosorbent assay was performed using commercial kits according to the manufacturer's instructions. Briefly, microplates were coated with capture antibodies, washed and blocked with blocking buffer. Test samples and standards were added and incubated, followed by washing and the addition of enzyme-labeled secondary antibodies, followed by incubation. After washing again, substrate mixture was added for color development, and absorbance was measured at 450 nm by an enzyme label instrument. The target concentration was calculated according to the corresponding standard curve.

[0028] 1.12 Isolation and processing of mouse prostate tissue Tumor tissue was obtained from a mouse and cut into pieces with surgical scissors. The cut tissue was placed in 0.25% trypsin-EDTA and digested in a 37°C water bath for 10 minutes. Then the tissue was transferred to DMEM containing 10% fetal bovine serum, 1 mg / ml collagenase type IV (Invitrogen) and 0.1 mg / ml DNase I (Invitrogen), and incubated at 37°C with gentle stirring for 2.5 hours. The digested tissue was centrifuged at 400 x g for 5 minutes, and the supernatant was discarded. After washing once with Hank's balanced salt solution, the tissue was incubated again in 0.25% trypsin-EDTA at 37°C for 10 minutes. Finally, the cells were resuspended in DMEM medium containing 10% fetal bovine serum and 0.4 mg / ml DNase I, and the digested tissue was filtered through a 40 micron filter to obtain a single cell suspension.

[0029] 1.13 Flow cytometry Cells were stained with fluorescein-conjugated antibodies against CD45 (BD Pharmingen™, San Jose) and CD8 (abcam, ab316778) as surface markers. After stimulation with PMA (50 ng / ml) and ionomycin (500 ng / ml) in the presence of Brefeldin A (BioLegend, San Diego), cells were fixed and permeabilized by a Fixation / Permeabilization Kit (Invitrogen, Waltham) to detect intracellular cytokines, followed by staining with IFN-γ (Affinity, DF6045) and granzyme B (Abcam, ab317458) antibodies. Data were collected by flow cytometry and analyzed by FlowJo software v10.

[0030] 2. Related results: 2.1 CRISPR / Cas9-mediated Mutp53 accelerates tumor progression and remodels the tumor microenvironment To investigate the function of Mutp53 in prostate cancer, we introduced Trp53 p.R245Q (a mouse homolog of human TP53 p.R248Q) into Myc-CaP cells using CRISPR / Cas9 genome editing technology. Figure 1 A and Figure 9 Compared with the control group tumors, the volume and endpoint weight of Mutp53 tumors in subcutaneous transplanted tumors were significantly increased. Figure 1 BD). This phenotype was also reproduced in the RM-1 model, with accelerated tumor growth and a larger tumor burden in the Mutp53 group. Figure 1 EG). KM analysis of an independent RM-1 cohort confirmed that mice carrying Mutp53 had reduced overall survival ( Figure 1 H; log-rank test p=0.0156).

[0031] To characterize genotype-related changes in the tumor microenvironment, we performed single-cell RNA sequencing on homologous tumors and annotated major lineages using classical markers. Figure 1 I). UMAP embedding reveals the expected partition structure ( Figure 1 J). Quantitative analysis of cell fractions showed that the number of fibroblasts in Mutp53 tumors was significantly reduced (p=0.0022), while the number of epithelial cells, endothelial cells, pericytes, and monocytes / macrophages was significantly increased. Figure 10 The differences in the number of T / NK cells and the number of T / NK cells were not statistically significant. Figure 1 K). Consistently, Ro / e (the ratio of observed to expected values) showed enrichment of fibroblasts and pericytes in WTp53 tumors, while Mutp53 tumors showed overexpression of myeloid and endothelial cell populations, with a moderate increase in T / NK cells. Figure 1 L). Multiplex immunofluorescence provided qualitative support, showing elevated signal intensity in Mutp53 tumors from CD4+FOXP3+ regulatory T cells, CD8+ T cells, and CD68+ / CD163+ macrophages, while CTLA-4 was slightly elevated. Figure 1 In summary, these data not only establish Trp53 p.R245Q as a driver of tumor growth, but also indicate that this genotype is associated with the remodeling of stromal and immune components in the tumor microenvironment.

[0032] 2.2 Mutp53 maintains proliferation by reprogramming epithelial state and metabolism To explore how Mutp53 affects epithelial heterogeneity and functional plasticity, we reclassified the epithelial cells into seven transcriptional states (Cluster0 to Cluster6, C0 to C6) Figure 2 A). This analysis showed that C0 and C2 were significantly expanded, while C3-C5 were depleted in Mutp53 tumors Figure 2 B). Similarly, Ro / e analysis showed that C0, C2 and C6 were enriched in Mutp53, while C3-C5 were more likely to be associated with WTp53 Figure 2 C). Functionally, C0 was characterized by extracellular matrix and adhesion programs (e.g., ECM organization, cell-matrix adhesion, focal adhesion), C2 by enhanced metal and calcium transport, high migration potential, and enrichment of steroid / terpenoid metabolism pathways, and C6 by interferon type I-driven antiviral and innate immune features Figure 2 D). Pseudo-time trajectory analysis placed C0, C2 and C6 in the terminal branch of the trajectory, which was consistent with the trend of epithelial cells developing towards higher malignant degree fate Figure 2 E). Meanwhile, metabolic scores (single-cell metabolic analysis, single-cell metabolomics) showed that the metabolic activity of arachidonic acid and fructose / mannose was significantly increased in Mutp53 epithelial cells Figure 2 F and Figure 11 ). LY3522348 or Glafenine selectively inhibited the proliferation of Mutp53 cells in CCK-8 Figure 2 G) and EdU detection Figure 2 H-I). Taken together, Mutp53 promoted the reprogramming of epithelial lineage, shifting towards adhesion, migration and antiviral states, while activating fructose and arachidonic acid metabolism to maintain tumor proliferation; importantly, targeting these activated metabolic pathways can reverse metabolic reprogramming, thus it is expected to be a potential strategy to combat Mutp53-driven tumor progression.

[0033] 2.3 Mutp53 tumors show stronger response to PD-1 blockade therapy by enhancing CD8+ cytotoxic program Immunosuppressive tumor microenvironment is often associated with poor response to anti-PD-1 therapy; however, clinical evidence suggests that Mutp53 tumor patients may instead benefit more from immune checkpoint blockade. To directly verify this hypothesis, we established subcutaneous transplantation and orthotopic transplantation RM-1 tumor models using WTp53 or Mutp53 cells Figure 3A). Consistent with previous studies, Mutp53 tumors exhibited faster growth rate than WT p53 tumors under basal conditions. In the subcutaneous model, PD-1 blockade significantly inhibited the progression of Mutp53 tumors, resulting in a significant reduction in tumor volume and endpoint weight compared to untreated controls (Fig. 1A). Figure 3 B-D). Similar patterns were observed in the orthotopic model, Mutp53 tumors also exhibited stronger basal growth capacity compared to WT p53 tumors, but responded more significantly to anti-PD-1 treatment, as evidenced by a more pronounced reduction in tumor burden (Fig. 1B). Figure 3 E-G).

[0034] In terms of mechanisms, flow cytometry analysis showed that Mutp53 tumors had a higher baseline proportion of CD8+GZMB+cytotoxic T cells, and further expanded upon PD-1 blockade treatment (Fig. 1F). Figure 3 H-I). Single-cell analysis of CD8+T cell subsets showed that CD8+, CD8+CD69+, CD8+GZMA+GZMB+, CD8+NKG7+, and CD4+CD8+T cells were significantly enriched in Mutp53 groups compared to WT p53 groups (Fig. 1H). Figure 3 J-K). AUCell analysis of intratumoral CD8+cells showed that cytotoxic programs were significantly upregulated in Mutp53 tumors (Fig. 1J). Figure 3 P-Q), while co-stimulatory, exhausted, and tissue-resident related features were not significantly different between genotypes (Fig. 1P). Figure 3 L-O, R-S). In particular, key effector genes such as Nkg7, Gzmb, and Slamf7 were significantly upregulated in Mutp53 tumors compared to WT p53 tumors, while IFN-g expression was not upregulated (Fig. 1L). Figure 3 T, and Figure 12 A). Multiplex immunofluorescence further confirmed these results, showing that in Mutp53 tumors, CD8+GZMB+T cell infiltration was increased both at baseline and after PD-1 blockade, while IFN-g expression was decreased (Fig. 1A). Figure 3 U, and Figure 12 B-C).

[0035] 2.4 Mutp53 enhances immunotherapy response by driving Cxcl10-mediated CD8+T cell recruitment To investigate the potential mechanisms of CD8+ T cell activation in Mutp53 tumors, we first used qRT-PCR to detect the expression of 15 previously confirmed immunochemokines associated with anti-PD-1 responses in prostate tumors derived from subcutaneous WTp53 and Mutp53 models (31, 32). Among these genes, Cxcl10, Cxcl1, Cxcl2, Cxcl12, Ccl2, and Ccl9 were significantly upregulated in Mutp53 tumors ( Figure 4 AB). Further analysis of the IMvigor210 urothelial carcinoma cohort treated with ICB revealed that Cxcl10 expression is highly correlated with multiple immune activation pathways, especially the IFN-γ signaling pathway and antigen presentation mechanism (APM). Figure 4 C), and both pathways are closely related to improved efficacy of immunotherapy. Consistent with the above findings, we confirmed by qRT-PCR and Western blotting that Cxcl10 overexpression exists in both WTp53 and Mutp53 tumor cell lines and tissues. Figure 4 DF). Transwell co-culture experiments showed that CD8+ T cells significantly enhanced the chemotactic migration ability of Mutp53-derived tumor cells. Figure 4 G). Clinical data show that patients with high intratumoral Cxcl10 expression significantly improved their response to immunotherapy. Figure 4 H).

[0036] To validate the function of this pathway in vivo, we pharmacologically blocked the Cxcl10-CXCR3 signaling pathway in WTp53 and Mutp53 tumor-bearing mice that had received PD-1 blockade therapy using a CXCR3 antagonist. Figure 4 I). Inhibition of this signaling axis reduced the therapeutic efficacy of PD-1 blockade in both models, with a particularly significant impact on Mutp53 tumors, specifically manifested in increased tumor volume and endpoint weight. Figure 4 (JL). In summary, these data indicate that Mutp53 enhances immunotherapy sensitivity by driving CD8+ T cell recruitment through Cxcl10, revealing a targetable chemokine axis that is crucial for maintaining anti-tumor immunity.

[0037] 2.5 Mutp53 promotes Cxcl10 transcription by enhancing H3K4me3 deposition and removing repressive histone markers. To clarify the mechanism of Cxcl10 upregulation in Mutp53 tumors, we first silenced the Trp53 gene using siRNA and observed a significant decrease in Cxcl10 expression, indicating that Mutp53 is involved in maintaining the transcription of Cxcl10 mRNA. Figure 5A). Potential p53 binding sites were identified in the Cxcl10 promoter region through JASPAR motif analysis. Figure 5 B). Consistent with its direct effects, CHIP-qPCR analysis showed that Mutp53 occupied a significantly higher proportion of the promoter region identified by primer 2 than WTp53. Figure 5 CD).

[0038] To assess the chromatin status of this locus, we detected histone markers using ChIP. The results showed that H3K4me3 enrichment was extremely low in WTp53 tumors for the region detected by primer 1, but significantly increased in Mutp53 tumors. Figure 5 E); For the detection region of primer 2, H3K4me3 enrichment was increased in Mutp53 tumors, while H3K27me3 and H3K36me3 levels were decreased ( Figure 5 F). Agarose gel electrophoresis results of the ChIP amplification products further validated the above enrichment pattern (F). Figure 5 G). In summary, these data indicate that, compared to WTp53, Mutp53 preferentially binds to the Cxcl10 promoter (primer 2), and the recruitment and activation mechanism involves increasing H3K4me3 and reducing repressive markers (H3K27me3, H3K36me3), thereby promoting RNA polymerase II-mediated Cxcl10 transcription. Figure 5 H).

[0039] 2.6 Mutp53 can promote the formation of lymphocyte aggregation characteristics without inducing the formation of an ordered tertiary lymphoid structure. To investigate whether Mutp53 affects immunological features associated with TLS, we first analyzed paired histopathological and genomic data from the TCGA cohort. The mutation frequency of TP53 was significantly higher in TLS+ tumors than in the corresponding TLS- tumors (17% vs. 10%). Figure 6 A), which provides population-level support for the association between TP53 mutations and a TLS-related immune environment. To validate this relationship in vivo, we performed multiplex immunofluorescence staining for CD3, CD20, and CD31 in a syngeneic tumor model (WTp53 vs. Mutp53, ± anti-PD-1 therapy). Although well-structured TLS was not observed, we detected significantly higher densities of CD3+, CD20+, and CD31+ cells in Mutp53 tumors compared to WTp53 tumors (A). Figure 6 (B–G). Notably, CD3+, CD20+, and CD31+ cell populations further increased after anti-PD-1 treatment. These data collectively suggest that Mutp53 promotes lymphocyte / angiogenesis.

[0040] Furthermore, given the known role of CAFs in forming physical and chemical barriers that impede T cell infiltration, we next assessed whether Mutp53 modulates CAF composition. Single-cell transcriptomic analysis segregated tumor-associated fibroblasts into myofibroblastic CAFs (myCAF), S100a10+inflammatory CAFs (iCAF), C4b+iCAF, and Cox5a+fibroblasts (fibro) Figure 7 A). The proportion of all four subpopulations was decreased in Mutp53 tumors compared to the proportion in WTp53 tumors ( Figure 7 B-C). Moreover, pseudotemporal trajectory analysis showed that myCAF was at the central branch node of fibroblast differentiation ( Figure 7 D-E), suggesting that Mutp53 can globally alter the developmental progression of CAFs. At the tissue level, multiplex immunofluorescence showed that the a-SMA+CAF area was decreased while CD8+T cell infiltration was increased in Mutp53 tumors. In contrast, in WTp53 tumors, CD8+T cells were often co-localized with a-SMA+CAF ( Figure 7 G), which is consistent with the previous model that CAFs restrict T cell entry into the tumor core. Taken together Figures 1-7 、 Figures 9-11 and the data in Tables S1-S2, Mutp53 enhances the efficacy of PD-1 blockade therapy by reprogramming the tumor microenvironment into an immune-permissive state. This state is characterized by attenuated fibroblast-driven exclusion and Cxcl10-mediated CD8+T cell recruitment. By coordinating the remodeling of epithelial, immune, and stromal cells, Mutp53 transforms an immunosuppressive tumor into one that is responsive to checkpoint inhibitors, providing a direct mechanistic basis for the increased sensitivity of p53-mutant prostate cancer to immune checkpoint blockade therapy.

Claims

1. Use of a PD-1 inhibitor in the manufacture of a medicament for treating prostate cancer with TP53 gene mutation.

2. Use according to claim 1, characterized in that, The medicament comprises: a therapeutically effective amount of a PD-1 inhibitor, and a pharmaceutically acceptable carrier auxiliary component.

3. Use according to claim 1, characterized in that, The TP53 gene mutation is any one of missense mutation, gene deletion, frameshift mutation, splice site mutation, truncation mutation.

4. The use according to claim 1, wherein The inhibitor is any one of PD-1 inhibitor, Glafenine inhibitor, LY3522348 inhibitor, AMG487 inhibitor.

Images