WO2023202223A1 - Réactif pour le traitement du cancer de la prostate neuroendocrinien et son utilisation - Google Patents

Réactif pour le traitement du cancer de la prostate neuroendocrinien et son utilisation Download PDF

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WO2023202223A1
WO2023202223A1 PCT/CN2023/078916 CN2023078916W WO2023202223A1 WO 2023202223 A1 WO2023202223 A1 WO 2023202223A1 CN 2023078916 W CN2023078916 W CN 2023078916W WO 2023202223 A1 WO2023202223 A1 WO 2023202223A1
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kit
foxa2
inhibitor
foxa1
gene
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PCT/CN2023/078916
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Chinese (zh)
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高栋
李飞
韩铭
张晔晗
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中国科学院分子细胞科学卓越创新中心
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the invention relates to the field of biomedicine, and specifically to the treatment of neuroendocrine prostate with tyrosine kinase receptor inhibitors.
  • Prostate cancer is the malignant tumor that ranks first in incidence and second in mortality among men in European and American countries (Siegel, RL, Miller, KD, Fuchs, HE, and Jemal, A. (2021). Cancer Statistics, 2021. CA Cancer J Clin 71, 7-33), and its incidence in China also shows an increasing trend year by year (Fu, ZT, Guo, XL, Zhang, SW, Zheng, RS, Zeng, HM, Chen, R., Wang, SM , Sun, KX, Wei, WW, and He, J. (2020). [Statistical analysis of incidence and mortality of prostate cancer in China, 2015]. Zhonghua Zhong Liu Za Zhi 42, 718-722.).
  • prostate cancer With the advancement of my country's modernization process, the Westernization of diet and the aging of the population, prostate cancer will develop into one of the important malignant tumors affecting the quality of life and health of Chinese men.
  • the normal prostate epithelium in adults is composed of luminal cells, basal cells and very rare neuroendocrine cells. Different from the composition of normal prostate epithelium, the pathological characteristic of primary prostate cancer is the massive expansion of luminal cells.
  • the androgen receptor (AR) binds to androgen and enters the nucleus to exert the gene regulatory function of a transcription factor, which is crucial for cell survival and proliferation (Huggins, C., and Hodges, CV (1972). Studies on prostatic cancer. I.
  • the present invention first provides the use of a reagent in preparing a drug for treating or preventing neuroendocrine prostate cancer.
  • the reagent includes one or more selected from the following:
  • FOXA2 inhibitors include inhibitory molecules that interfere with the transcription and/or expression of the gene encoding the FOXA2 protein, or downregulate the activity of the FOXA2 protein.
  • the inhibitory molecules use proteins or their encoding genes or transcripts as inhibition targets.
  • the inhibitory molecule is selected from the group consisting of: (1) small molecule compounds, antisense nucleic acids, microRNA, siRNA, shRNA, dsRNA, sgRNA, LncRNA, specific antibodies or ligands, or combinations thereof , and (2) a nucleic acid construct capable of expressing or forming (1).
  • the inhibitory molecule is siRNA or a construct thereof that uses the FOXA2 protein coding gene or its transcript as an inhibition target.
  • the FOXA2 inhibitor is an agent, such as sgRNA, that knocks out or knocks down the FOXA2 gene using a technology selected from ZFN, TALEN, and CRISPR.
  • the FOXA1 promoter is an agent that upregulates FOXA1 expression or activity.
  • the FOXA1 promoter is selected from the group consisting of small molecule compounds, nucleic acid molecules, or combinations thereof.
  • the nucleic acid molecule is a nucleic acid construct containing a FOXA1 coding sequence.
  • the amino acid sequence of FOXA1 is selected from one or more of the following: (a) a polypeptide having the sequence shown in SEQ ID NO: 1 or 2; (b) SEQ ID NO: 1 or 2 The sequence shown is formed by the substitution, deletion or addition of one or more (such as 1-20; preferably 1-10; more preferably 1-5) amino acid residues, and has the function of (a) polypeptide. (a) A derived polypeptide; or (c) A polypeptide composed of (c) having more than 90% (preferably 93%; more preferably 95% or 98%) homology with the polypeptide sequence of (a) and having the function of (a) polypeptide. a) Derivatized polypeptides.
  • the KIT response signaling pathway inhibitor includes a KIT inhibitor.
  • KIT inhibitors include inhibitory molecules that interfere with the transcription and/or expression of the gene encoding the KIT protein, or downregulate the activity of the KIT protein.
  • the inhibitory molecules use proteins or their encoding genes or transcripts as inhibition targets.
  • the inhibitory molecule is selected from the group consisting of: (1) small molecule compounds, antisense nucleic acids, microRNA, siRNA, shRNA, dsRNA, sgRNA, LncRNA, specific antibodies or ligands, or combinations thereof , and (2) a nucleic acid construct capable of expressing or forming (1).
  • the inhibitory molecule is siRNA or a construct thereof that uses the KIT protein encoding gene or its transcript as an inhibition target.
  • the KIT inhibitor is an agent, such as sgRNA, that knocks out or knocks down the KIT gene using a technology selected from ZFN, TALEN, and CRISPR.
  • the KIT inhibitor is a drug targeting other tumors harboring KIT mutants.
  • the KIT inhibitor is a tyrosine kinase receptor inhibitor, preferably Imatinib, Sorafenib, Sunitinib, and Cabozantin Cabozantinib.
  • the present invention also provides a pharmaceutical composition, including active ingredients and pharmaceutically acceptable excipients, and the active ingredients include one or more selected from the following:
  • FOXA2 inhibitors include inhibitory molecules that interfere with the transcription and/or expression of the gene encoding the FOXA2 protein, or downregulate the activity of the FOXA2 protein.
  • the inhibitory molecules use proteins or their encoding genes or transcripts as inhibition targets.
  • the pH The manufactured molecule is selected from the group consisting of: (1) small molecule compounds, antisense nucleic acids, microRNA, siRNA, shRNA, dsRNA, sgRNA, LncRNA, specific antibodies or ligands, or combinations thereof, and (2) capable of expressing or forming (1 ) nucleic acid constructs.
  • the inhibitory molecule is siRNA or a construct thereof that uses the FOXA2 protein coding gene or its transcript as an inhibition target.
  • the FOXA1 promoter is an agent that upregulates FOXA1 expression or activity.
  • the FOXA1 promoter is selected from the group consisting of small molecule compounds, nucleic acid molecules, or combinations thereof.
  • the nucleic acid molecule is a nucleic acid construct containing a FOXA1 coding sequence.
  • the amino acid sequence of FOXA1 is selected from one or more of the following: (a) a polypeptide having the sequence shown in SEQ ID NO: 1 or 2; (b) SEQ ID NO: The sequence shown in 1 or 2 is formed by the substitution, deletion or addition of one or more (such as 1-20; preferably 1-10; more preferably 1-5) amino acid residues, and has (a) A polypeptide derived from (a) that has polypeptide function; or (c) has more than 90% (preferably 93%; more preferably 95% or 98%) homology with the polypeptide sequence of (a) and has a polypeptide of (a) Functional polypeptide derived from (a).
  • the KIT response signaling pathway inhibitor includes a KIT inhibitor.
  • KIT inhibitors include inhibitory molecules that interfere with the transcription and/or expression of the gene encoding the KIT protein, or downregulate the activity of the KIT protein.
  • the inhibitory molecules use proteins or their encoding genes or transcripts as inhibition targets.
  • the inhibitory molecule is selected from the group consisting of: (1) small molecule compounds, antisense nucleic acids, microRNA, siRNA, shRNA, dsRNA, sgRNA, LncRNA, specific antibodies or ligands, or combinations thereof , and (2) a nucleic acid construct capable of expressing or forming (1).
  • the inhibitory molecule is siRNA or a construct thereof that uses the KIT protein encoding gene or its transcript as an inhibition target.
  • the KIT inhibitor is a drug targeting other tumors harboring KIT mutants.
  • the KIT inhibitor is a tyrosine kinase receptor inhibitor, preferably Imatinib, Sorafenib and Sunitinib.
  • the present invention also provides the FOXA1 gene or protein, the FOXA2 gene or protein or the genes or proteins of the KIT response signaling pathway as targets for screening drugs for treating or preventing neuroendocrine prostate cancer in animals. for clinical application in neuroendocrine prostate cancer.
  • the animal is a mammal, preferably a rat, rabbit or human.
  • the gene of the KIT responsive signaling pathway includes a KIT gene.
  • the drug is a FOXA1 promoter, a FOXA2 inhibitor, or a KIT inhibitor.
  • This article also provides applications of reagents for detecting FOXA1 genes or proteins, FOXA2 genes or proteins, or genes or proteins of the KIT response signaling pathway in preparing kits for diagnosing neuroendocrine prostate cancer.
  • This article also provides a detection kit, which contains reagents for detecting FOXA1 genes or proteins, FOXA2 genes or proteins, or genes or proteins of the KIT response signaling pathway, such as primers, probes, or specific proteins for detecting genes. sexual antibodies.
  • the detection kit further includes nucleic acid detection reagents or immunological detection reagents, such as reagents required for PCR and reagents required for ELISA.
  • Figure 1 shows the single-cell multi-omics landscape of NEPC progression.
  • A Immunofluorescence staining images of Syp, Trp63, Krt8 and Ar in TPPRC tumors at different stages of NEPC progression. Scale bar is 50 ⁇ m.
  • B Schematic diagram of experimental design for single-cell multi-omics sequencing of TPPRC tumors.
  • C Schematic diagram of single-cell multi-omics sequencing of ATAC and RNA from the same cell.
  • D UMAP diagram based on RNA, ATAC or WNN respectively. Cell annotation was performed based on representative lineage markers from WNN analysis.
  • E Heatmap of RNA expression levels of representative marker genes in the cell annotation group.
  • F Dot plot of RNA expression levels of representative marker genes in the cell annotation population.
  • G Heatmap plot of chromatin accessibility levels of representative marker genes in the cell annotation population.
  • H Visualization of chromatin accessibility for representative marker gene loci in each cell population.
  • Figure 2 shows the analysis of cellular heterogeneity during the progression of NEPC.
  • A UMAP map of prostate lumen and neuroendocrine cells calculated by WNN analysis.
  • B Feature diagram of Ar RNA expression levels in all cell populations.
  • C Feature plot of RNA expression levels of Chga in all cell populations.
  • D Diagram of cellular composition at different stages of NEPC progression.
  • E Heatmap annotating the RNA expression levels of the top 10 marker genes in each cell population. Representative marker genes for each cell population are labeled with different colors.
  • F Pathway enrichment for each cell population and its associated network.
  • Figure 3 illustrates the identification of Foxa2 as a pioneer transcription factor regulating the neuroendocrine lineage.
  • A WnnUMAP plot used for pseudo-temporal analysis.
  • B Violin plot of pseudotime for 5 cell populations.
  • C Violin plot of pseudo-time at different stages of NEPC progression.
  • D WnnUMAP plot of two tumor progression trajectories.
  • E WnnUMAP plot of process trajectories for lineage 1.
  • F Process trajectory wnnUMAP map of lineage 2.
  • G Heat map of dynamic changes in chromatin accessibility during pseudo-time progression of NEPC.
  • H RNA expression heat map of genes that dynamically change during pseudo-time progression of NEPC.
  • Q Box plot of RNA expression levels of Foxa2 in WCM.
  • R Box plot of RNA expression levels of Foxa2 in UW.
  • S Feature plot of the RNA expression levels of Foxa2 and Foxa1 based on UMAP, indicating the mutually exclusive expression patterns of Foxa2 and Foxa1. The red dashed line represents luminal cells, and the blue dashed line represents neuroendocrine cells.
  • T Violin plot of Foxa1 RNA expression levels in all cell types.
  • U Violin plot of Foxa2 RNA expression levels in all cell types. P values were calculated based on two-tailed tests.
  • Figure 4 shows that Foxa2 regulates neuroendocrine differentiation and promotes Kit-mediated cell proliferation.
  • A Heatmap plot based on Foxa1 ChIP intensity at early and late stages of NEPC progression.
  • B Based on NEPC Heatmap plot of Foxa2 ChIP intensity at early and late stages of progression.
  • C The number of target genes bound by Foxa1 was visualized in the four groups respectively. IGV plots show peak intensities for representative target gene sites.
  • Sox2 and Fkbp5 are target genes of Foxa1. Sox2 is a NEPC characteristic gene, and its expression is negatively correlated with Foxa1 expression.
  • Fkbp5 is an ARPC characteristic gene, and its expression is positively correlated with Foxa1 expression.
  • Syt1 and Trim36 are target genes of Foxa2.
  • Syt1 is a NEPC characteristic gene, and its expression is positively correlated with Foxa2 expression.
  • Trim36 is an ARPC characteristic gene, and its expression is negatively correlated with Foxa2 expression.
  • E Venn plot of the number of Foxa1-specific peaks, Foxa2-specific peaks, and overlapping peaks.
  • (N) QRT-PCR results of relative RNA expression levels of Foxa2 in ARPC cells overexpressing PCDH-EV or PCDH-Foxa2. Two-tailed t test, mean ⁇ SEM, n 3.
  • (O) QRT-PCR results of relative RNA expression levels of Kit in ARPC cells overexpressing PCDH-EV or PCDH-Foxa2. Two-tailed t test, mean ⁇ SEM, n 3.
  • Figure 5 shows cell-cell interactions revealing that the Kit signaling pathway is a neuroendocrine-specific communication method.
  • A Circle plot of interaction strength between each pair of cell types during NEPC progression. Line thickness indicates interaction strength, and arrows indicate the cell type that receives the signal.
  • B Dot plot of communication intensity across all cell types. Dot size indicates communication strength.
  • C Alluvial plot of signal pattern received by target cells.
  • D Dot plot of signal pattern received by target cells. Red dotted boxes mark neuroendocrine-specific pathways.
  • E Dot plot of neuroendocrine-specific reception signal patterns. color Represents signal contribution.
  • F Kit signaling pathway network display diagram. Arrows indicate target cells.
  • G Kit Signal Network. The edge width represents the communication probability.
  • Figure 6 shows that clinical-grade inhibitors of the Kit signaling pathway significantly slowed NEPC progression.
  • B NEPC organoid formation assay results on days 0, 2, and 4 after drug treatment. Scale bar is 50 ⁇ m.
  • G Growth curves of NEPC tumors in mice treated with PBS, Imatinib (50mg/kg), sorafenib (50mg/kg), sunitinib (40mg/kg) and enzalutamide (10mg/kg) respectively.
  • One-way ANOVA and Tukey's test, mean ⁇ SEM, n 8-16.
  • H Box plot of CHGA and Kit RNA expression levels in the three data sets of FHCRC, WCM and UW.
  • I Dot plot of Kit expression levels.
  • J Heat map of RNA expression levels of characteristic marker genes and NEPC and ARPC scores calculated by GSVA.
  • K Cell viability assay results of human NEPC organoid ST88.
  • treatment and “treatment” and the like refer to any action that provides benefits to patients suffering from NEPC, including improving the condition by reducing or inhibiting at least one symptom and delaying disease progression.
  • prevention refers to actions that provide benefits to patients at risk of NEPC, including avoiding discomfort or progression of the disease or reducing one or more symptoms of the disease if it occurs. Family history may place this patient at risk.
  • the "subject” or “patient” involved in the treatment, treatment, and diagnosis described herein is an animal at risk of suffering from NEPC, usually a mammal, such as a mouse (such as a mouse), a rabbit, or a human.
  • Kit signaling pathway is crucial in mediating cell interactions carried out by neuroendocrine tumor cells, and that tyrosine kinase receptor Kit inhibitors can significantly slow down the tumor progression of NEPC. Therefore, Kit can be used as a drug target for NEPC treatment.
  • Kit can be used as a drug target for NEPC treatment.
  • Foxa2 directly regulates Kit expression and regulates the differentiation of the neuroendocrine lineage; the gene expression levels and motif activities of Foxa2 and Foxa1 show a negative correlation. As NEPC tumors progress, the DNA binding activity of Foxa1 decreases.
  • the present invention involves using FOXA2 (mouse: NCBI Entrez Gene: 15376, amino acid sequence as shown in SEQ ID NO: 3; human: NCBI Entrez Gene: 3170, amino acid sequence as shown in SEQ ID NO: 4) as a target therapy or Prevent NEPC.
  • FOXA2 molecular biological formula 2
  • the present invention relates to the use of a FOXA2 inhibitor in the preparation of a medicament for preventing, slowing down, treating, or regressing neuroendocrine prostate cancer.
  • FOXA2 inhibitors include inhibitory molecules that interfere with the transcription and/or expression of the gene encoding the FOXA2 protein, or downregulate the activity of the FOXA2 protein.
  • the inhibitory molecules use proteins or their encoding genes or transcripts as inhibition targets.
  • the inhibitory molecule is selected from the group consisting of: (1) small molecule compounds, antisense nucleic acids, microRNA, siRNA, shRNA, dsRNA, sgRNA, LncRNA, specific antibodies or ligands, or combinations thereof , and (2) a nucleic acid construct capable of expressing or forming (1).
  • the inhibitory molecule is siRNA or a construct thereof that uses the FOXA2 protein coding gene or its transcript as an inhibition target.
  • FOXA2 inhibitors can also be knockout or Reagents that knock down the FOXA2 gene, such as sgRNA.
  • the present invention involves using FOXA1 (mouse: NCBI Entrez Gene: 15375, amino acid sequence as shown in SEQ ID NO: 1; human: NCBI Entrez Gene: 3169, amino acid sequence as shown in SEQ ID NO: 2) as a target therapy or Prevent NEPC.
  • FOXA1 molecular genetics
  • the present invention relates to the use of a FOXA1 promoter in the preparation of a medicament for preventing, slowing down, treating, or regressing neuroendocrine prostate cancer.
  • the FOXA1 promoter is an agent that upregulates FOXA1 expression or activity.
  • the FOXA1 promoter is selected from the group consisting of small molecule compounds, nucleic acid molecules, or combinations thereof.
  • the nucleic acid molecule is a nucleic acid construct containing a FOXA1 coding sequence.
  • the amino acid sequence of FOXA1 is selected from one or more of the following: (a) a polypeptide with the sequence shown in SEQ ID NO: 1; (b) the sequence shown in SEQ ID NO: 1 through one or more (such as 1- A polypeptide derived from (a) formed by substitution, deletion or addition of 20; preferably 1-10; more preferably 1-5) amino acid residues and having (a) polypeptide function; or (c) A polypeptide derived from (a) that has more than 90% (preferably 93%; more preferably 95% or 98%) homology with the polypeptide sequence of (a) and has the function of the polypeptide of (a).
  • the present invention relates to the treatment or prevention of NEPC by targeting KIT response signaling pathway genes and proteins. Specifically, the present invention relates to the use of KIT response signaling pathway inhibitors in the preparation of medicaments for preventing, slowing down, treating neuroendocrine prostate cancer, or regressing the disease.
  • the present invention provides a method of treating neuroendocrine prostate cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an agent that targets the expression or activity of a KIT response-related signaling pathway protein.
  • the KIT response signaling pathway inhibitor can be a KIT protein (mouse: NCBI Entrez Gene: 16590, the amino acid sequence is shown in SEQ ID NO: 5; human: NCBI Entrez Gene: 3815, the amino acid sequence is shown in SEQ ID NO: 6) Inhibitors.
  • KIT inhibitors include inhibitory molecules that interfere with the transcription and/or expression of the gene encoding the KIT protein, or downregulate the activity of the KIT protein.
  • the inhibitory molecules use proteins or their encoding genes or transcripts as inhibition targets.
  • the inhibitory molecule is selected from the group consisting of: (1) small molecule compounds, antisense nucleic acids, microRNA, siRNA, shRNA, dsRNA, sgRNA, LncRNA, specific antibodies or ligands, or combinations thereof, and (2) a nucleic acid construct capable of expressing or forming (1).
  • the inhibitory molecule is siRNA or a construct thereof that uses the KIT protein encoding gene or its transcript as an inhibition target.
  • KIT inhibitors are agents that can also knock out or knock down the KIT gene using technologies selected from ZFN, TALEN and CRISPR, such as sgRNA.
  • KIT inhibitors can also be drugs targeting other tumors with KIT mutations.
  • the KIT inhibitor is a tyrosine kinase receptor inhibitor, preferably Imatinib, Sorafenib and Sunitinib.
  • the present invention also provides the application of FOXA1 gene or protein, FOXA2 gene or protein or KIT response signaling pathway gene (such as KIT gene) or protein as a target in screening drugs for treating or preventing neuroendocrine prostate cancer, or as a molecular indicator.
  • the drug is a FOXA1 promoter, a FOXA2 inhibitor or a KIT inhibitor.
  • the present invention also provides a method for screening drugs, including: (1) contacting the candidate drug with FOXA1 protein, FOXA2 protein or KIT response signaling pathway protein (such as KIT) or nucleic acid molecules encoding them or systems containing them, and (2) detecting Changes in the expression or activity of the protein or its encoding nucleic acid molecule may indicate a candidate drug as a target drug. If the candidate drug can increase the expression or activity of FOXA1 protein, FOXA2 protein or KIT response signaling pathway protein (such as KIT) or its encoding nucleic acid molecule, it indicates that the candidate drug is a potential substance for preventing or treating NEPC.
  • step (1) includes: adding the candidate substance to the system of FOXA1 protein, FOXA2 protein or KIT protein in the test group; and/or step (2) includes: detecting FOXA1 protein in the system of the test group , the expression or activity of FOXA2 protein or KIT protein, and compared with a control group, wherein the control group is a system expressing FOXA1 protein, FOXA2 protein or KIT protein without adding the candidate substance. If the expression of FOXA1 protein in the test group is statistically higher (preferably significantly higher, such as more than 20% higher, preferably more than 50% higher; more preferably more than 80% higher) than the control group, it indicates that the candidate It is a potential substance for preventing or treating NEPC.
  • FOXA2 protein or KIT protein in the test group is statistically lower than (preferably significantly lower than, such as more than 20% lower, preferably more than 50% lower; more preferably more than 80% lower) the control group, it means that This candidate is a potential substance for the prevention or treatment of NEPC.
  • the present invention also provides a pharmaceutical composition, which contains an effective amount of a FOXA2 inhibitor, a FOXA1 promoter, or a KIT response signaling pathway inhibitor as an active ingredient and a pharmaceutically acceptable excipient combined with the active ingredient.
  • the pharmaceutical composition of the present invention includes one or more active ingredients and one or more pharmaceutically acceptable excipients.
  • pharmaceutically acceptable excipients refers to excipients used in the administration of therapeutic agents, including various excipients, diluents and carriers. Such excipients include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • Pharmaceutically acceptable carriers can be solid or liquid.
  • Suitable pharmaceutically acceptable carriers include, but are not limited to, magnesium carbonate, magnesium stearate, talc, sugar, lactose, sesame oils, synthetic fatty acid esters such as ethyl oleate or triglycerides or polyglycerides. Ethylene glycol 400, hydrogenated castor oil and cyclodextrin, etc.
  • the pharmaceutical preparation is matched to the mode of administration, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical composition is preferably manufactured under sterile conditions.
  • the medicine or pharmaceutical composition of the present invention can be in any suitable dosage form, including powder, tablet, dispersible granule, capsule, cachet, suppository, solution, suspension, transdermal agent, injection, sustained release agents and emulsions, etc.
  • the prostates of TPPRC mice were isolated and minced with scissors.
  • the isolated prostate cells were resuspended in red blood cell lysis solution (Miltenyi Biotec, Cat. No. 130-094-183), and after incubation at room temperature for 2 minutes, dead cells were removed using the Dead Cell Removal Kit (Miltenyi Biotec, Cat. No. 130-090-101). cells, and cell counting was performed with a Countess II FL automated cell counter (Thermo Fisher Scientific). Cells were then lysed on ice for 5 minutes. The isolated nuclei are transferred and loaded onto the Chromium Next GEM chip. The chip containing the prostate cells was then loaded onto the controller. Subsequent library construction was performed according to the manufacturer's instructions.
  • the library establishment steps for ATAC-seq are carried out with reference to the Omni-ATAC method (Corces et al., 2017).
  • the specific steps are as follows: pre-cool the centrifuge to 4°C in advance, prepare Wash buffer and Lysis buffer, and
  • the buffer configuration is as follows.
  • Imatinib (S1026, Selleck) dissolved in PBS, sorafenib (Selleck, S1040) and sunitinib (Selleck, S1042) dissolved in DMSO. Mix 10,000 NEPC cells and Matrigel evenly and inoculate them into a 96-well suspension plate. After Matrigel solidifies, add 100ul of culture medium. The next day, add 100 ⁇ L of prostate organoid culture medium containing placebo or different concentrations of inhibitors. Organoid growth experiments were performed on ST88 and BM1 cells with a starting cell number of 4500. After drug treatment for a certain period of time, cell viability was measured using CellTiter Glo 3D (Promega).
  • Imatinib (Selleck, S1026) was dissolved in PBS at a concentration of 50 mg/150 ⁇ L before use. Enzalutamide was dissolved in a solution containing 1% carboxymethyl cellulose, 0.1% Tween 80, and 5% DMSO. TPPRC tumors were transplanted into SCID mice by subcutaneous injection.
  • mice were randomly assigned to various treatment groups and treated with imatinib (50mg/kg) or PBS by intraperitoneal injection twice a day; sorafenib was treated with Enzalutamide (10 mg/kg) and sunitinib (Selleck, S7781) (40 mg/kg) are administered as a once-daily intraperitoneal injection (Selleck, S7397) (50 mg/kg). Administration was administered by gavage feeding.
  • Example 1 Lineage plasticity in prostate cancer progression
  • mice model In order to explore the specific mechanism of lineage plasticity transformation in prostate cancer progression, we first established a mouse model with Pten, Trp53 and Rb1 deletion driven by induction of the luminal cell-specific gene Tmprss2 CreERT2 .
  • tdTomato was placed downstream of the endogenous gene Chga promoter and translationally fixed termination element (LSL), and this mouse model was called TPPRC (Tmprss2 CreERT2/+ ; Pten flox/flox ; p53 flox/flox ; Rb1 flox/flox ; Chga LSL-tdTomato/+ ).
  • TPPRC Tmprss2 CreERT2/+ ; Pten flox/flox ; p53 flox/flox ; Rb1 flox/flox ; Chga LSL-tdTomato/+ .
  • This mouse model not only ensures that NEPC originates from luminal cells, but also the expression of tdTomato can directly indicate neuroendoc
  • Prostate tumors in TPPRC mice were analyzed at different time points, including 2 weeks, 1 month, 2.5 months, 3.5 months, 4.5 months and 6 months after tamoxifen injection.
  • the appearance of Syp + /tdTomato + neuroendocrine cancer cells was observed 1 month after tamoxifen injection, and as expected, the percentage of neuroendocrine cancer cells gradually increased over time (Fig. 1, a).
  • the simultaneous expression pattern of tdTomato and Syp demonstrated that tdTomato can accurately instruct neuroendocrine cancer cell differentiation (Fig. 1, a).
  • RNA expression and chromatin accessibility data were integrated using weighted nearest neighbor analysis (WNN), and 13 cell populations from different lineages were identified.
  • WNN weighted nearest neighbor analysis
  • RNA expression pattern of Ar and Chga clearly showed that cell populations 1 and 2 had characteristics of luminal cells, while cell populations 3, 4, and 5 had characteristics of neuroendocrine cells (Fig. 2, bc), which are consistent with tumor progression.
  • the changes in cell composition were consistent (Fig. 2, d).
  • Example 3 Foxa2 is a pioneer transcription factor regulating NEPC lineage plasticity
  • Example 4 Foxa2 regulates the differentiation of prostate adenocarcinoma into neuroendocrine cancer and promotes Kit-mediated cell proliferation
  • Example 5 Kit pathway is a neuroendocrine cancer cell-specific communication method
  • c-Kit encoded by the Kit gene is mainly activated by the secreted stem cell factor encoded by Kitl for further conduction of intracellular signals. Therefore, this signaling interaction through soluble factors and membrane-bound receptors is critical for regulating a variety of biological processes including cell growth or death, migration, and differentiation.
  • Example 6 Inhibiting the tyrosine kinase receptor Kit pathway can be an effective means to potentially treat NEPC
  • Imatinib also known as "Gleevec”
  • Gleevec is a tyrosine kinase inhibitor and is currently the only drug recommended by the National Comprehensive Cancer Network clinical practice guidelines for patients with Kit mutations. Melanoma Drugs.
  • prostate tumors derived from TPPRC mice injected with tamoxifen for 4.5 months were digested and used for the construction of primary tumor organoids.
  • Kit inhibitors were transplanted into SCID mice via subcutaneous injection. Consistent with the results of in vitro organoid culture experiments, all three Kit inhibitors consistently demonstrated significant effects in inhibiting NEPC tumor progression (Figure 6, g).
  • Kit has a similar function in human NEPC.
  • NEPC had significantly higher Kit expression levels in all three data sets ( Figure 6, h).
  • Kit expression levels were more likely to exhibit neuroendocrine differentiation (Fig. 6, i).
  • imatinib could limit the growth of human NEPC tumor cells.
  • the NEPC organoid ST88 constructed earlier in the laboratory was used to evaluate the effect of imatinib, and it was found that imatinib significantly inhibited the growth of ST88 ( Figure 6, j).
  • Kit inhibitors can effectively slow down the progression of NEPC and have certain tumor type specificity.
  • tyrosine kinase receptor Kit signaling pathway plays a specific and important role in mediating cell interactions carried out by neuroendocrine tumor cells, and Kit is proposed as a drug target for NEPC treatment.
  • Kit's ligand Kitl is widely expressed in various types of cells such as epithelial cells, mesenchymal cells, and immune cells, while Kit is specifically and highly expressed in neuroendocrine tumor cells, further confirming that Kit Great potential as specific drug targets.
  • tyrosine kinase receptor Kit inhibitors including imatinib, sorafenib, and sunitinib
  • imatinib, sorafenib, and sunitinib can It very significantly slows down the tumor progression of NEPC, and the inhibitory effect has certain tumor type selectivity.
  • imatinib, sorafenib, and sunitinib have all been approved by the FDA for the treatment of various other types of tumors, so these inhibitors have clear pharmacokinetic characteristics and high safety profiles. Its various advantages largely ensure that it can quickly enter clinical practice for evaluation of efficacy through the "new use of old drugs" approach.

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Abstract

La présente invention concerne un réactif pour le traitement du cancer de la prostate neuroendocrinien et son utilisation. La présente invention concerne spécifiquement l'utilisation du réactif dans la préparation d'un médicament pour le traitement ou la prévention du cancer de la prostate neuroendocrinien. Le réactif comprend un ou plusieurs composants choisis dans le groupe constitué par (1) un inhibiteur de FOXA2, (2) un promoteur FOXA1, et (3) un inhibiteur de la voie de signalisation sensible à KIT. La présente invention identifie une nouvelle cible pour le traitement ou la prévention du cancer de la prostate neuroendocrinien.
PCT/CN2023/078916 2022-04-22 2023-03-01 Réactif pour le traitement du cancer de la prostate neuroendocrinien et son utilisation WO2023202223A1 (fr)

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CN108403659A (zh) * 2018-03-01 2018-08-17 中国科学院过程工程研究所 一种硬乳液纳微球及其制备方法和应用
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