WO2024050861A1 - Utilisation d'un inhibiteur de runx2 dans la préparation d'un médicament de lutte contre des tumeurs phyllodes malignes du sein - Google Patents

Utilisation d'un inhibiteur de runx2 dans la préparation d'un médicament de lutte contre des tumeurs phyllodes malignes du sein Download PDF

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WO2024050861A1
WO2024050861A1 PCT/CN2022/118895 CN2022118895W WO2024050861A1 WO 2024050861 A1 WO2024050861 A1 WO 2024050861A1 CN 2022118895 W CN2022118895 W CN 2022118895W WO 2024050861 A1 WO2024050861 A1 WO 2024050861A1
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runx2
breast
cells
malignant
tumors
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Chinese (zh)
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聂燕
宋尔卫
雷蓉
黄红颜
贺诗施
艾力菲热***
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中山大学孙逸仙纪念医院
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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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/14—Drugs for genital or sexual disorders; Contraceptives for lactation disorders, e.g. galactorrhoea

Definitions

  • the invention relates to the field of medical biotechnology, and specifically relates to the application of RUNX2 inhibitors in the preparation of drugs against malignant phyllodes tumors of the breast.
  • Phyllodes tumor is a rare fibroepithelial tumor, accounting for 0.3% to 1% of all breast tumors. According to existing literature research, borderline and malignant phyllodes tumors not only grow rapidly, but are also prone to local recurrence and distant metastasis. Blood metastasis is the most common way of metastasis, and lung and bone are the most common metastasis sites. The recurrence rate of malignant phyllodes tumors of the breast is as high as 53.1%, and the metastasis rate is as high as 43.1%. Once recurrence and metastasis occur, the patient will die within a short period of time. The mortality rate of malignant phyllodes tumors is approximately 16.3%.
  • breast phyllodes tumors The treatment of breast phyllodes tumors is mainly surgical. Malignant phyllodes tumors have poor clinical prognosis due to their high recurrence rate and high metastasis rate. Unlike breast cancer, adjuvant treatments such as chemotherapy and radiotherapy are not effective after surgery for malignant phyllodes tumors of the breast. There are currently no studies reporting clinical benefit from targeted therapy and immunotherapy. Moreover, because phyllodes tumors of the breast are relatively rare, there is a lack of large-scale clinical data and basic research, especially research on specific molecular markers and treatment targets, and no effective progress has been made, further resulting in the lack of research or screening. Progress in effective treatments has stalled.
  • RUNX2 is an important member of the RUNX family of transcription factors and is named because it contains a runt domain.
  • the biological role of RUNX2 is mainly as a specific transcription factor for osteogenic differentiation, regulating the transcription of genes such as type I collagen, osteomodulin, osteocalcin, col1a1, col1a2, bone sialoprotein (BSP) and fibronectin. It plays an important role in the formation and differentiation of osteoblasts, differentiation and maturation of chondrocytes, the formation and resorption of osteoclasts, and the synthesis of bone matrix proteins.
  • BSP bone sialoprotein
  • RUNX2 is related to the biological behavior of malignant phyllodes tumors of the breast.
  • the present invention discloses the technical application of RUNX2 as a biological behavioral marker and drug target of malignant phyllodes tumors of the breast.
  • RUNX2 is highly expressed in malignant breast phyllodes tumor cells and promotes the progression of malignant breast phyllodes tumors. Knocking down the expression of RUNX2 can inhibit the proliferation, migration, and invasion of malignant phyllodes tumors of the breast.
  • RUNX2 can be used as a marker and drug target for malignant phyllodes tumors of the breast, and has high application value in the auxiliary diagnosis, screening and preparation of targeted therapeutic drugs for breast phyllodes tumors. The following applications are thus exposed:
  • RUNX2 as a marker in screening or preparing drugs against malignant breast phyllodes tumors.
  • detection of RUNX2 expression in test tumor cells can be used as a means to evaluate drug efficacy.
  • RUNX2 as a drug target in screening or preparing drugs against malignant phyllodes tumors of the breast.
  • Drugs targeting RUNX2 as a therapeutic target can be prioritized for screening as potential drugs against breast malignant phyllodes tumors.
  • the drug CADD522 inhibits the binding of RUNX2 to DNA.
  • the invention further discloses the application of the RUNX2 inhibitor CADD522 in the preparation of drugs against malignant phyllodes tumors of the breast.
  • the present invention has the following beneficial effects:
  • the present invention discloses for the first time that the overexpression of RUNX2 in breast malignant phyllodes tumor specimens is significantly related to the biological behavior of breast malignant phyllodes tumors. Knocking down the expression of RUNX2 can inhibit the proliferation, migration, and invasion of breast malignant phyllodes tumors. It is believed that RUNX2 can be used as a marker and therapeutic target for screening drugs against malignant phyllodes tumors of the breast, and has high application value in the screening and preparation of drugs targeting malignant phyllodes tumors of the breast.
  • CADD522 can significantly inhibit the proliferation, migration, and invasion of breast malignant phyllodes tumor cells in vitro, and can significantly inhibit the growth of tumors in vivo.
  • CADD522 has great potential clinical application value in the development of targeted therapeutic drugs for breast phyllodes tumors.
  • Figure 1 A is a histogram of RUNX2 mRNA expression levels in benign and malignant breast phyllodes tumor tissues; B is a protein immunoblot gel imaging image of RUNX2 protein expression test in benign and malignant breast phyllodes tumor tissues; C is benign , RUNX2 expression levels in paraffin sections of malignant phyllodes tumors and RUNX2 expression levels in paraffin sections of multiple recurrences of tumor tissue from the same patient.
  • Figure 2 A is a graphical representation of the test results of knocking down the RUNX2 gene of the breast malignant phyllodes tumor cell line SYSH-MPT-01 (i.e., the cell line HJP-0320, disclosed in the patent application with publication number CN111019898A) and changing the cell proliferation ability.
  • SYSH-MPT-01 i.e., the cell line HJP-0320, disclosed in the patent application with publication number CN111019898A
  • B knocks down the RUNX2 gene of the breast malignant phyllodes tumor cell line SYSH-MPT-01, and the results of the test on changes in cell colony formation ability
  • C shows the knockdown of the RUNX2 gene of the breast malignant phyllodes tumor cell line SYSH-MPT-01, Graphical representation of cell cycle change test results
  • D is a graphic representation of the knockdown of the RUNX2 gene in the breast malignant phyllodes tumor cell line SYSH-MPT-01, and the results of a cell migration and invasion ability change test
  • E is a graphic representation of the knockdown of the breast malignant phyllodes tumor cell line Illustration of the test results of the RUNX2 gene of SYSH-MPT-01 and the changes in cell collagen contraction ability.
  • FIG. 3 A is a graphical representation of the test results of changes in cell proliferation ability overexpressing RUNX2 in the benign breast phyllodes tumor cell line SYSH-BPT-01 (i.e., the cell line GLK-1010, disclosed in the patent application with publication number CN111019897A) ; B is a graphical representation of the test results of the overexpression of RUNX2 in the benign breast phyllodes tumor cell line SYSH-BPT-01, and the changes in the clonogenic ability of the cells; C is the overexpression of RUNX2 in the benign breast phyllodes tumor cell line SYSH-BPT-01 RUNX2, a graphical representation of the test results of cell cycle changes; D is a graphical representation of the test results of overexpression of RUNX2 in the benign breast phyllodes tumor cell line SYSH-BPT-01, and the changes in cell migration and invasion capabilities; E is a graphic representation of the test results of the benign breast phyllodes tumor RUNX2 is overex
  • Figure 4 is a graphical representation of the results of a trial on the impact of RUNX2 on the prognosis of patients with breast phyllodes tumors.
  • Figure 5 is a graphical representation of the results of a test to knock down RUNX2 in malignant phyllodes tumor cells on subcutaneous tumor formation ability and tumor growth; A is the change in tumor volume at the corresponding time point, and B is a picture of the tumors in each group.
  • FIG. 6 is a diagram showing the effect of CADD522 on the malignant phyllodes tumor cell lines SYSH-BPT-01 and SYSH-MPT-02 (i.e., the cell line LJ-0429, disclosed in the patent application with publication number CN111019899A);
  • A is CADD522 The survival rate curve acting on the malignant phyllodes tumor cell line, illustrating the determination of the half inhibitory concentration (IC50) of CADD522 on the malignant phyllodes tumor cell line;
  • B is the growth of the malignant phyllodes tumor cell line SYSH-MPT-01 inhibited by CADD522 , showing a dose- and time-dependent diagram;
  • C is a diagram showing the effect of CADD522 on the migration and invasion ability of breast malignant phyllodes tumor cells.
  • Figure 7 is a diagram showing the effect of CADD522 on malignant phyllodes tumors in vivo.
  • A is the inhibition of tumor growth by intraperitoneal injection of CADD522 in the PDX model of malignant phyllodes tumors, and the changes in tumor volume at corresponding time points.
  • B is the pictures of tumors in each group.
  • Example 1 Analysis of RUNX 2 expression profile of breast phyllodes tumors
  • RUNX2-specific primers to perform fluorescence quantitative PCR to detect the expression levels of RUNX2 mRNA in different types of breast phyllodes tumor tissues.
  • Reverse transcription to synthesize cDNA Reverse transcription to synthesize cDNA: Add 1 ⁇ g of template RNA and 4 ⁇ l of reverse transcriptase SuperScript II mix (containing Buffer, dNTP, HiScript II reverse transcriptase, RNase, Random primers/Oligo dT) in the PCR tube.
  • Real-time quantitative PCR amplification Dilute the template cDNA 3 times and mix well for later use. Each experimental group was set up with 3 parallel tubes. Reaction system: 1 ⁇ l cDNA, 5 ⁇ l SYBR green dye, 0.3 ⁇ l forward primer, 0.3 ⁇ l reverse primer, 3.4 ⁇ l ddH 2 O, centrifuge and mix. Reaction conditions: 95°C for 5min, 95°C for 30 seconds, 55°C-60°C (depending on the annealing temperature) for 30s, a total of 40 cycles. GAPDH was used as an internal reference to analyze the relative transcription levels of genes. The primer sequences are as follows:
  • Example 2 Effects of knocking down RUNX2 on the proliferation, migration, invasion, cell cycle and collagen contraction ability of malignant phyllodes tumor cells
  • siRNA The sequence of siRNA is as follows:
  • Example 6 Effect of RUNX2 small molecule inhibitor CADD522 on IC50, proliferation, migration and invasion of malignant phyllodes tumor cells
  • the RUNX2 small molecule inhibitor CADD522 inhibits the migration and invasion of malignant phyllodes tumor cells
  • the RUNX2 small molecule inhibitor CADD522 inhibits PDX tumor growth

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Abstract

L'invention concerne l'utilisation de RUNX2 en tant que marqueur et que cible de traitement dans le criblage et la préparation d'un médicament de lutte contre des tumeurs phyllodes malignes du sein, ainsi que l'utilisation de l'inhibiteur CAD522 de RUNX2 dans la préparation d'un médicament de lutte contre des tumeurs phyllodes malignes du sein. Il a été divulgué que la surexpression de RUNX2 est liée aux comportements biologiques des tumeurs phyllodes malignes du sein, et l'inactivation de l'expression de RUNX2 permet d'inhiber la prolifération, la migration et l'invasion des tumeurs phyllodes malignes du sein. Une preuve expérimentale a montré que l'inhibiteur CAD522 de petites molécules, spécifique de RUNX2, peut inhiber la prolifération, la migration et l'invasion de cellules tumorales phyllodes malignes du sein in vitro et peut inhiber une croissance tumorale in vivo.
PCT/CN2022/118895 2022-09-08 2022-09-15 Utilisation d'un inhibiteur de runx2 dans la préparation d'un médicament de lutte contre des tumeurs phyllodes malignes du sein WO2024050861A1 (fr)

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CN202211092785.0 2022-09-08
CN202211092785.0A CN115851932A (zh) 2022-09-08 2022-09-08 Runx2抑制剂在制备抗乳腺恶性叶状肿瘤药物中的应用

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016149667A1 (fr) * 2015-03-19 2016-09-22 University Of Maryland , Baltimore Inhibiteurs du facteur de transcription runx2 et utilisations associées
WO2021048292A1 (fr) * 2019-09-11 2021-03-18 INSERM (Institut National de la Santé et de la Recherche Médicale) Méthodes et compositions pour le traitement d'un mélanome
US20220065865A1 (en) * 2018-12-21 2022-03-03 Uea Enterprises Limited Methods of treatment and diagnosis of tumours

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016149667A1 (fr) * 2015-03-19 2016-09-22 University Of Maryland , Baltimore Inhibiteurs du facteur de transcription runx2 et utilisations associées
US20220065865A1 (en) * 2018-12-21 2022-03-03 Uea Enterprises Limited Methods of treatment and diagnosis of tumours
WO2021048292A1 (fr) * 2019-09-11 2021-03-18 INSERM (Institut National de la Santé et de la Recherche Médicale) Méthodes et compositions pour le traitement d'un mélanome

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KIM MYOUNG SOOK, GERNAPUDI RAMKISHORE, CEDEÑO YESSENIA CEDEÑO, POLSTER BRIAN M., MARTINEZ RAMON, SHAPIRO PAUL, KESARI SANTOSH, NUR: "Targeting breast cancer metabolism with a novel inhibitor of mitochondrial ATP synthesis", ONCOTARGET, IMPACT JOURNALS LLC, UNITED STATES, vol. 11, no. 43, 27 October 2020 (2020-10-27), United States , pages 3863 - 3885, XP093146967, ISSN: 1949-2553, DOI: 10.18632/oncotarget.27743 *
KIM MYOUNG SOOK, GERNAPUDI RAMKISHORE, CHOI EUN YONG, LAPIDUS RENA G., PASSANITI ANTONINO: "Characterization of CADD522, a small molecule that inhibits RUNX2-DNA binding and exhibits antitumor activity", ONCOTARGET, IMPACT JOURNALS LLC, UNITED STATES, vol. 8, no. 41, 19 September 2017 (2017-09-19), United States , pages 70916 - 70940, XP093134573, ISSN: 1949-2553, DOI: 10.18632/oncotarget.20200 *
ZUO ZHONGKUN, YE FEI, LIU ZIRU, HUANG JIANGSHENG, GONG YI: "MicroRNA‑153 inhibits cell proliferation, migration, invasion and epithelial‑mesenchymal transition in breast cancer via direct targeting of RUNX2", EXPERIMENTAL AND THERAPEUTIC MEDICINE, SPANDIDOS PUBLICATIONS, GR, vol. 18, 5 April 2019 (2019-04-05), GR , XP093146968, ISSN: 1792-0981, DOI: 10.3892/etm.2019.7470 *

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