Patent Grant
11931401
This application claims the benefit of priority from Korean Patent Application No. 10-2020-0049341, filed on Apr. 23, 2020, and Korean Patent Application No. 10-2020-0184526, filed on Dec. 28, 2020, the contents of each of which are incorporated herein by reference.
The present invention relates to a pharmaceutical composition comprising a Runx3 modified protein as an active ingredient for prevention or treatment of cancer.
Research on the development of targeted cancer therapy is focused on strategies to control cancer cells by inhibiting the function of an oncogene or activating the function of a tumor suppressor gene. Abnormal activation of K-Ras function by mutation of K-Ras among the oncogenes is known as one of the major causes of human cancer. The mutation of K-Ras is also observed in lung cancer, and it is known that the mutation of K-Ras is observed in about 35% of lung adenocarcinoma. Thus, in order to treat cancer caused by the activation of K-Ras function, studies have been conducted on a method of treating cancer by inhibiting the function of K-Ras. However, a strategy that directly inhibits the function of K-Ras has not been developed as a successful anticancer drug because it causes serious damage to normal cells. Therefore, instead of suppressing the function of an oncogene, a strategy of activating the inhibited function of a tumor suppressor gene is receiving attention. Therefore, instead of a strategy for inhibiting the function of an oncogene, a strategy for activating the inhibited function of a tumor suppressor gene is attracting attention.
The said tumor suppressor gene refers to a nucleotide sequence that can be expressed in a target cell to suppress a tumor phenotype or induce apoptosis. The tumor suppressor genes identified so far include sPD-1, VHL, MMAC1, DCC, p53, NF1, WT1, Rb, BRCA1 and BRCA2. Among them, it has been reported that p53 or Rb gene is frequently inhibited in its function in K-Ras mutant cancers. Whether it is possible to treat K-Ras mutant cancer through the repair of the suppressor gene has become a subject of great interest in the field of anticancer agent development research. Accordingly, there have been attempts to treat K-Ras mutant lung adenocarcinoma by recovering the function of p53 gene, which is a representative tumor suppressor gene, but it was not successful because early lung adenocarcinoma was not cured (Feldser, D. M. et al., Nature, 468: 572-575, 2010, Junttila, M. R. et al., Nature, 468: 567-571, 2010). In addition, it was found that K-Ras mutant lung cancer could not be cured through the recovery of Rb gene function (Walter, D. M. et al. Nature 2019). The above results indicate that even if the function of the tumor suppressor gene is simply restored, the therapeutic effect on the already-onset cancer does not appear, because the early stage cancer rapidly develops into a malignant cancer (Berns A., Nature, 468:519-520, 2010). There have been no reports of successful treatment of K-Ras mutant lung cancer through the activation of a tumor suppressor gene.
It has been reported that the function of Runx3 gene as a tumor suppressor gene is inhibited in K-Ras mutant cancers (RUNX3 Protects against Oncogenic KRAS. (2013). Cancer Discovery, 4(1), 14-14), and that the activity of Runx3 gene is inhibited in lung adenocarcinoma caused by the mutation of K-Ras (Lee, K. S., Lee, Y. S., Lee, J. M., Ito, K., Cinghu, S., Kim, J. H., Bae, S. C. Oncogene, 29(23): 3349-61, 2010).
Runx3, a transcription factor that binds to DNA, plays a crucial role in lineage determination (Ito, Y., Bae, S. C. & Chuang, L. S. The RUNX family: developmental regulators in cancer. Nat. Rev. Cancer 15, 81-95 (2015)). Deletion of Runx3 in the mouse lung leads to the development of lung adenomas and accelerates the progression to adenocarcinoma (ADCs) induced by K-Ras.
Thus, the present inventors have completed the present invention by confirming that the modified protein in which the 356th serine of Runx3 is substituted with alanine has an increased activity of maintaining the complex with Brd2 compared to the wild-type Runx3, and the apoptosis effect is improved in various cancer cell lines compared to the wild-type Runx3.
It is an object of the present invention to provide a pharmaceutical composition comprising a Runx3 modified protein as an active ingredient for prevention or treatment of cancer.
To achieve the above object, the present invention provides a pharmaceutical composition for prevention or treatment of cancer, comprising a modified protein in which the 356th serine of Runx3 (Runt-related transcription factor 3) is substituted with a hydrophobic amino acid, a polynucleotide coding thereof, a vector carrying the polynucleotide, or a virus or cell transformed with the vector as an active ingredient.
In the present invention, it was confirmed that the modified protein in which the 356th serine of Runx3 is substituted with alanine has an increased activity of maintaining the complex with Brd2 by more than 10 times compared to the wild-type Runx3, and the apoptosis effect is improved in various cancer cell lines compared to the wild-type Runx3. Therefore, the modified protein in which the 356th serine of Runx3 is substituted with an amino acid that cannot be phosphorylated by a kinase of the present invention, the polynucleotide coding thereof, the vector carrying the polynucleotide, or the virus or cell transformed with the vector can be used as a therapeutic agent for various cancers.
The Sequence Listing is submitted as an ASCII text file in the form of the file name “Sequence.txt” (˜22 kb), which was created on Apr. 22, 2021, and which is incorporated by reference herein.
Hereinafter, the present invention is described in detail.
The present invention provides a pharmaceutical composition for prevention or treatment of cancer, comprising a modified protein in which the 356th serine of Runx3 (Runt-related transcription factor 3) is substituted with an amino acid that cannot be phosphorylated by a kinase, a polynucleotide coding thereof, a vector carrying the polynucleotide, or a virus or cell transformed with the vector as an active ingredient.
The cancer is solid cancer.
The solid cancer can be one or more selected from the group consisting of lung cancer, pancreatic cancer, liver cancer and stomach cancer, but not always limited thereto.
The amino acid that cannot be phosphorylated by a kinase can be one or more selected from the group consisting of alanine (A), isoleucine (I), leucine (L) and valine (V), but not always limited thereto.
Runx3 (Runt-related transcription factor 3) gene is one of the Runt family genes consisting of Runx1, Runx2 and Runx3. The Runt family genes play an important role in normal development and oncogenesis, and they function as transcriptional regulators of the Smad family, a downstream factor that mediates TGF-β and its signaling. Runx1 plays an important role in mammalian hematopoiesis, Runx2 plays an important role in bone formation, and Runx3 is mainly expressed in granular gastric mucosal cells, and plays a role in inhibiting cell differentiation of gastric epithelium. These three genes are located at loci of chromosomes 1p, 6p and 21q, of which Runx3 gene is located at 1p36. 11-1p36. 13. The Runx3 locus is one of the sites that are lost in a variety of cancers or affected by hemizygous defects. In addition, Runx3 has been found to be inactivated in various types of cancer, and it is gaining spotlight as a new target for the development of anticancer agents. As such, Runx3 is known to act as a tumor suppressor gene that suppresses the formation of cancer, and plays an important role in the restriction-point, which determines the fate of cell division and death, and induces cell division or apoptosis depending on the situation (Lee et al., Nat Commun. 2019; 10(1): Runx3 regulates cell cycle-dependent chromatin dynamics by functioning as a pioneer factor of the restriction-point). When a K-Ras oncogene mutation occurs in lung epithelial cells, Runx3 kills cancer cells by contributing to determining apoptosis fate at the restriction-point (Lee et al., Nat Commun. 2019; 10(1)).
A Runx3 protein refers to a Runt-related transcription factor 3 related to the Runt family expressed by the Runx3 gene.
The Runx3 protein can be composed of the amino acid sequence represented by SEQ. ID. NO: 1 or SEQ. ID. NO: 2.
The Runx3 protein can be derived from humans or animals.
The Runx3 protein can be synthesized by the conventional chemical synthesis method in the art (W. H. Freeman and Co., Proteins; structures and molecular principles, 1983), or can be prepared by the conventional genetic engineering method (Maniatis et al., Molecular Cloning: A laboratory Manual, Cold Spring Harbor laboratory, 1982; Sambrook et al., Molecular Cloning: A Laboratory Manual et al.).
The Runx3 protein can be a variant of an amino acid sequence having a different sequence by deletion, insertion or substitution of amino acid residues, or a combination thereof within a range that does not affect the function of the protein. Amino acid exchanges in proteins that do not totally alter the activity of the molecule are informed in the art. In some cases, the amino acid can be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation or farnesylation. Accordingly, the present invention can include a peptide having an amino acid sequence substantially identical to that of a protein composed of the amino acid sequence represented by SEQ. ID. NO: 1 or SEQ. ID. NO: 2, and variants or fragments thereof. The substantially identical protein can have homology to the protein of the present invention by 80% or more, particularly 90% or more, and more particularly 95% or more.
The vector including the polynucleotide encoding the modified protein in which the 356th serine of Runx3 protein is substituted with an amino acid that cannot be phosphorylated by a kinase can be linear DNA or plasmid DNA.
The polynucleotide encoding the Runx3 protein can be composed of the amino acid sequence represented by SEQ. ID. NO: 3 or SEQ. ID. NO: 4.
The vector refers to a transport mediator for introducing the polynucleotide encoding the modified protein in which the 356th serine of Runx3 protein is substituted with an amino acid that cannot be phosphorylated by a kinase of the present invention into a subject to be treated, and can include a promoter suitable for expression in a subject to be treated, an enhancer, and a polynucleotide encoding the Runx3 protein, a transcription termination site, and the like. The promoter can be a specific organ and tissue specific promoter, and can include a replication origin so as to proliferate in the organ and tissue.
BRD2 (Bromodomain-containing protein 2) is a factor that acts as a signaling mediator in the nucleus. It is widely expressed in mammalian cells, and plays an important role in cell cycle regulation and transcriptional regulation.
The BRD2 binds to the acetylated Runx3.
The BRD2 is composed of BD1 and BD2.
The bromodomain 1 (BD1) of the BRD2 binds to the lysine residues 94 and 171 of Runx3.
The bromodomain 2 (BD2) of the BRD2 binds to the lysine residue 5 of the acetylated histone 4, the lysine residue 12 of histone 4, and the lysine residue 14 of histone 3.
When the complex is formed, cell death occurs.
In addition, the complex is formed upon receiving mitogenic stimulation.
The complex contributes to the determination of restriction point (R-point).
The virus transformed by the vector can be any one selected from the group consisting of retrovirus, adenovirus, herpes simplex virus and lentivirus, but not always limited thereto.
In the case of the vector containing the polynucleotide, it is preferably to contain 0.05 to 500 mg, and more preferably to contain 0.1 to 300 mg. In the case of the recombinant virus containing the polynucleotide encoding the modified protein in which the 356th serine of Runx3 protein is substituted with an amino acid that cannot be phosphorylated by a kinase, it is preferably to contain 103 to 1012 IU (10 to 1010 PFU), and more preferably to contain 105 to 1010 IU.
The recombinant virus is preferably adenovirus. Adeno-associated virus (AAV) is unsuitable as a delivery vehicle for cancer treatment because its gene expression rate or expression speed is lower than that of adenovirus. Adenovirus is suitable for the delivery of the modified protein according to the present invention to the human body because the transferred gene is expressed in adenovirus more than 3 weeks faster than in adeno-associated virus (HUMAN GENE THERAPY 15:405-413.), and the phenomenon of lowering the gene transfer efficiency due to the immune response is less in the adenovirus than in the adeno-associated virus (World J Gastroenterol. 2016 Jan. 7; 22(1):326-37).
The number of viruses for treatment can be represented by the number of viral particles including the vector genome or the number of infectable viruses. That is, since about 1% of the virus particles are the effective number of viruses that can actually be infected, IU (infection unit) or PFU (plaque forming unit) is used to indicate this.
The cell transformed by the vector can be bacterium.
The bacterium can be non-pathogenic or non-toxic, and can be Listeria, Shigella, Salmonella, or E. coli. By introducing the vector into bacteria, DNA of a gene included in the vector can be mass-replicated or proteins can be mass-produced.
The vector according to the present invention can be introduced into cells using a method known in the art. For example, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun, and other known methods for introducing nucleic acids into cells can be used to introduce the vector into cells, but not always limited thereto (Wu et al., J. Bio. Chem., 267:963-967, 1992; Wu and Wu, J. Bio. Chem., 263:14621-14624, 1988).
In the case of the cells transformed with the vector containing the polynucleotide, it is preferably to contain 103 to 108 cells, and more preferably to contain 104 to 107 cells.
The pharmaceutical composition for prevention or treatment of cancer, comprising a modified protein in which the 356th serine of Runx3 (Runt-related transcription factor 3) is substituted with an amino acid that cannot be phosphorylated by a kinase, a polynucleotide coding thereof, a vector carrying the polynucleotide, or a virus or cell transformed with the vector as an active ingredient of the present invention can be administered parenterally during clinical administration.
The effective dose of the composition per 1 kg of body weight is 0.05 to 12.5 mg/kg for the vector, 107 to 1011 virus particles (105 to 109 IU)/kg for the recombinant virus, and 103 to 106 cells/kg for the cell. Preferably, the dose is 0.1 to 10 mg/kg for the vector, 108 to 1010 virus particles (106 to 108 IU)/kg for the recombinant virus, and 102 to 105 cells/kg for the cell. The composition can be administered 2 to 3 times a day. The composition as described above is not always limited thereto, and can vary depending on the conditions of a patient and the degree of onset of a disease.
The pharmaceutical composition according to the present invention may contain 10 to 95 weight % of a vector containing a Runx3 protein, a polynucleotide coding thereof, a vector carrying the polynucleotide, or a virus or cell transformed with the vector, which is an active ingredient, based on the total weight of the composition. In addition, the pharmaceutical composition of the present invention can include, in addition to the active ingredient, one or more effective ingredients having the same or similar function to the active ingredient.
In preferred embodiments of the present invention, the present inventors confirmed that phosphorylation did not occur in the modified protein in which the 356th amino acid of Runx3 is substituted with another amino acid (
Therefore, the modified protein in which the 356th serine of Runx3 (Runt-related transcription factor 3) is substituted with an amino acid that cannot be phosphorylated by a kinase, the polynucleotide coding thereof, the vector carrying the polynucleotide, or the virus or cell transformed with the vector can be used as a therapeutic agent for various cancers.
Hereinafter, the present invention will be described in detail by the following examples and experimental examples.
However, the following examples and experimental examples are only for illustrating the present invention, and the contents of the present invention are not limited thereto.
<Experimental Methods>
1. Cell Line Preparation
HEK293 cells (ATCC, Manassas, VA, USA) were maintained in DMEM medium (Gibco BRL, Thermo Fisher Scientific, MA, USA, MA) supplemented with 10% fetal bovine serum (Gibco BRL) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA).
H460 cells (ATCC, Manassas, VA, USA) and H460 stable cells were maintained in RPMI 1640 medium (Gibco BRL) supplemented with 10% fetal bovine serum (Gibco BRL) and 1% penicillin/streptomycin (Invitrogen). MKN28 cells (ATCC, Manassas, VA, USA) and PANC1 cells (ATCC, Manassas, VA, USA) were maintained in DMEM medium (Gibco BRL) supplemented with 10% fetal bovine serum (Gibco BRL) and penicillin/streptomycin (Invitrogen). All the cell lines were cultured in a 37° C., 5% CO2 incubator.
2. Introduction of Runx3 and Runx3 S356A
Each cell line was cultured in a 10 cm culture dish (5×105 cells) for 2 days and then approximately 2×106 cells of each cell line was infected with 1×108 VP of adenovirus or adenovirus Runx3 or adenovirus Runx3 S356A. After 48 hours, the cell death rate was measured by flow cytometry.
3. DNA Transfection, Immunoprecipitation (IP) and Immunoblotting (IB)
Transient transfection was performed in all cell lines using lipofectamine plus reagent and lipofectamine (Invitrogen). Cell lysates were incubated with an appropriate monoclonal or polyclonal antibody (2 μg of antibody/500 μg of lysate sample) at 4° C. for 3 hours, followed by incubation with protein G-Sepharose beads (Amersham Pharmacia Biotech, Piscataway, NJ, USA). For the detection of endogenous proteins at 4° C. for 1 hour, the lysate was incubated with an appropriate monoclonal or polyclonal antibody (1:1000˜1:3000) at 4° C. for 6 to 12 hours, and then protein G-Sepharose beads (Amersham Pharmacia Biotech) were heated at 4° C. for 3 hours. The immune precipitate was digested on an SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was blocked, immunoblotted with an appropriate antibody, treated with ECL solution (Amersham Pharmacia Biotech), and visualized in Amersham™ Imager 600 (GE Healthcare, Chicago, IL, USA).
4. Antibody
The antibody targeting RUNX3 (5G4) (Cat #ab40278) was obtained from Abcam (Cambridge, UK), and the antibody was diluted 1:3000. BRD2 (M01; 1:1000; Cat #H00006046-M01, Abnova, Taipei City, Taiwan) was used for immunoblotting and immunoprecipitation.
5. Flow Cytometry
Cells were harvested and processed using FITC-Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, CA, USA) and propidium iodide DNA staining protocol. Apoptosis and cell cycle were analyzed by flow cytometry on a BD FACS caliber machine (BD Biosciences). All data were analyzed using FlowJo software (https://www.flowjo.com).
In the polynucleotide encoding the Runx3 protein registered in Genebank, mutant recombination was performed in the animal cell expression vector pCS4-3flag-RUNX3 using the primer sets listed in Table 1 including EcoRI-XhoI cleavage sites at both ends to induce mutations in the codon encoding the amino acid sequence of the 356th serine.
Particularly, pCS4-flag-RUNX3 plasmid was digested with EcoRI/XhoI restriction enzyme and polymerase chain reaction (PCR) was performed with each Primer-F/Primer-M-R (Resulting Insert: F) and Primer-M-F/Primer-R (Resulting Insert: R) using RUNX3 Insert as a template. The PCR product was purified, and the secondary polymerase chain reaction was performed with F and R templates, Primer-F and Primer-R. The RUNX3 S356A PCR product obtained by the secondary polymerase chain reaction was purified. RUNX3 S356A and pCS4-flag-Vector were digested with EcoRI/XhoI restriction enzyme, gel-extracted, ligated, and transfected. Only single colony was cultured in 2 ml and of LB medium, and the cloned plasmid was separated and purified to confirm the nucleotide sequence.
RUNX3 phosphorylation in serine, the 356th amino acid of Runx3 protein, was measured by immunoprecipitation (IP) and immunoblotting (IB) in the same manner as described in Experimental Method 3 above.
As a result, as shown in
The possibility of phosphorylation of the 356th amino acid of the prepared mutant was verified using a phosphorylation level prediction tool (Phospho.elm:http://phospho.elm.eu.org/pELMB lastSearch.html) and NETPhos 3.1 (cbs.dtu.dk/services/NetPhos/). As a result, as shown in Table 2 and
The above results indicate that when the 356th amino acid of Runx3 was substituted with another amino acid, the phosphorylation of Runx3 was suppressed, and thus the time for maintaining the binding with BRD2 was increased.
The modified protein in which the 356th amino acid of Runx3 is substituted with alanine blocks the physical binding between Runx3 and CDK4, thereby inhibiting the phosphorylation of the 356th serine of Runx3 by CDK4. By inhibiting the conversion of the Rpa-Rx3-AC complex including Runx3 to the Rpa-Rx3-TR complex, the function of maintaining the anticancer activity of Runx3 is improved. Thus, the following experiment was performed to compare the time for the modified protein in which the 356th amino acid of Runx3 is substituted with alanine to form a complex by binding to Brd2 protein with the wild-type Runx3.
Particularly, the antibody targeting RUNX3 (5G4) (Cat #ab40278) was obtained from Abcam (Cambridge, UK), and the antibody was diluted 1:3000. BRD2 (M01; 1:1000; Cat #H00006046-M01, Abnova, Taipei City, Taiwan) was used for immunoblotting and immunoprecipitation. Transient transfection was performed in all cell lines using lipofectamine plus reagent and lipofectamine (Invitrogen). Cell lysates were incubated with an appropriate monoclonal or polyclonal antibody (2 μg of antibody/500 μg of lysate sample) at 4° C. for 3 hours, followed by incubation with protein G-Sepharose beads (Amersham Pharmacia Biotech, Piscataway, N.J., USA). For the detection of endogenous proteins at 4° C. for 1 hour, the lysate was incubated with an appropriate monoclonal or polyclonal antibody (1:1000˜1:3000) at 4° C. for 6 to 12 hours, and then protein G-Sepharose beads (Amersham Pharmacia Biotech) were heated at 4° C. for 3 hours. The immune precipitate was digested on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was blocked, immunoblotted with an appropriate antibody, treated with ECL solution (Amersham Pharmacia Biotech), and visualized in Amersham™ Imager 600 (GE Healthcare, Chicago, IL, USA).
As a result, as shown in
It was confirmed through flow cytometry that the Runx3 S356A modified protein improved the maintenance efficacy of the Runx3-Brd2 complex, and the apoptosis ability thereof was improved in various cancer cell lines compared to the wild-type Runx3.
Particularly, each cell line prepared according to Experimental Method 1 above was harvested and processed using FITC-Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, Calif., USA) and propidium iodide DNA staining protocol. Apoptosis and cell cycle were analyzed by flow cytometry on a BD FACS caliber machine (BD Biosciences). All data were analyzed using FlowJo software (https://www.flowjo.com).
As a result, as shown in
Western blotting was performed as described in Experimental Method 3 to confirm the expression of Runx3 protein in adenovirus expressing the Runx3 S356A modified protein and the wild-type Runx3.
As a result, as shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2020-0049341 | Apr 2020 | KR | national |
| 10-2020-0184526 | Dec 2020 | KR | national |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20080261883 | Bae | Oct 2008 | A1 |
| 20120128642 | Teumer et al. | May 2012 | A1 |
| 20150336967 | Czardybon et al. | Nov 2015 | A1 |
| 20180002393 | Bancel | Jan 2018 | A1 |
| Number | Date | Country |
|---|---|---|
| 10-1994957 | Jun 2019 | KR |
| WO2013151672 | Oct 2013 | WO |
| Entry |
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| Number | Date | Country | |
|---|---|---|---|
| 20210330740 A1 | Oct 2021 | US |
