KR102710867B1 - A pharmaceutical composition for enhancing the therapeutic effect of melanoma comprising an oligodendrocyte transcription factor 2 inhibitor as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for enhancing the metastasis and therapeutic effect of melanoma, and more specifically, it has been confirmed that when a composition containing a transcription factor Olig2 (Oligodendrocyte transcription factor 2) inhibitor is administered in combination with dabrafenib, the melanoma prevention or therapeutic effect of dabrafenib is significantly enhanced, and since the pharmaceutical composition of the present invention can effectively treat melanoma even when a lower dose of dabrafenib is used than before, it can be free from the problems of toxicity and side effects.

Description

{A pharmaceutical composition for enhancing the therapeutic effect of melanoma comprising an oligodendrocyte transcription factor 2 inhibitor as an active ingredient}

The present invention relates to a pharmaceutical composition for enhancing the therapeutic effect of melanoma, and more specifically, to a composition for enhancing the therapeutic effect of dabrafenib for melanoma, which comprises an inhibitor of the expression or activity of the transcription factor Olig2 (Oligodendrocyte transcription factor 2).

Due to recent lifestyles such as increased outdoor activities and the increased influx of ultraviolet rays due to the destruction of the ozone layer caused by environmental pollution, opportunities for exposure to various harmful substances or ultraviolet rays are becoming more frequent, and as the average human life expectancy increases, the number of elderly people who accumulate a lot of ultraviolet rays is increasing, and the number of patients related to skin cancer is continuously increasing worldwide. Skin cancer is a malignant skin tumor, and the representative types are basal cell carcinoma, squamous cell carcinoma, and melanoma. Among them, melanoma can occur in any part where melanocytes exist, such as the skin, eyeballs, and brain and spinal cord, and unlike other skin cancers, it is known to be a highly malignant tumor with a high rate of metastasis to other organs in the early stages of occurrence and easy recurrence.

The main metastatic site of melanoma is the skin other than the primary lesion, but it can also invade other organs such as lymph nodes, bones, lungs, liver, and spleen. In particular, melanoma has a high resistance to conventional chemotherapy and radiation, so it is an incurable disease with no specific treatment method at present when it progresses beyond the initial stage or relapses.

Transcription factor Olig2 is a transcription factor expressed in the central nervous system during embryonic development, and is known to regulate oligodendrocyte differentiation and cell cycle progression, and to promote the growth of brain tumors. Transcription factor Olig2 has been confirmed to be overexpressed in lung cancer, breast cancer, melanoma, and other cells in addition to oligodendroglioma (PCT application number: 10-2017-7027034). However, there has been no specific study on the effect of combining transcription factor Olig2 with existing treatments on the efficacy of existing treatments.

Against this background, the inventors of the present invention conducted research efforts to discover a substance for treating melanoma, and as a result, they confirmed that when an inhibitor of the transcription factor Olig2 (Oligodendrocyte transcription factor 2, hereinafter referred to as Olig2) and dabrafenib, known as a targeted treatment for melanoma, are used in combination, the growth of melanoma cells is inhibited more effectively than when dabrafenib is treated alone. Accordingly, they confirmed that an Olig2 inhibitor can enhance the therapeutic effect of dabrafenib on melanoma and also suppress the recurrence of melanoma patients administered dabrafenib, thereby completing the present invention.

Accordingly, the present invention aims to provide a pharmaceutical composition for preventing or treating melanoma, comprising a BRAF inhibitor and an inhibitor of expression or activity of the Olig2 (Oligodendrocyte transcription factor 2) gene or protein.

In addition, another object of the present invention is to provide a pharmaceutical composition for enhancing the effect of preventing or treating melanoma when used in combination with a BRAF inhibitor, characterized in that the composition comprises an inhibitor of the expression or activity of the transcription factor Olig2 (Oligodendrocyte transcription factor 2) as an active ingredient.

However, the technical problems to be achieved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

To achieve the above purpose, the present invention provides a pharmaceutical composition for preventing or treating melanoma; or preventing or treating metastasis of melanoma, comprising a BRAF inhibitor and an inhibitor of expression or activity of the Olig2 (Oligodendrocyte transcription factor 2) gene or protein.

In one embodiment of the present invention, the BRAF inhibitor may be dabrafenib.

In addition, the present invention provides a pharmaceutical composition for preventing or treating melanoma in combination with a BRAF inhibitor; or for enhancing the effect of preventing or treating metastasis of melanoma, characterized in that the composition comprises an inhibitor of the expression or activity of transcription factor Olig2 (Oligodendrocyte transcription factor 2) as an active ingredient.

In one embodiment of the present invention, the BRAF inhibitor may be dabrafenib.

In another embodiment of the present invention, the transcription factor Olig2 (Oligodendrocyte transcription factor 2) inhibitor may be any one selected from the group consisting of an antisense oligonucleotide, small interfering RNA (siRNA), short hairpin RNA (shRNA), and ribozyme specific to the transcription factor Olig2 (Oligodendrocyte transcription factor 2).

In another embodiment of the present invention, the activity inhibitor of the transcription factor Olig2 (Oligodendrocyte transcription factor 2) protein may be any one selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to the transcription factor Olig2 (Oligodendrocyte transcription factor 2) protein.

In another embodiment of the present invention, the combination may be administered simultaneously, separately or sequentially.

According to the present invention, it was confirmed that the melanoma therapeutic effect of dabrafenib is enhanced when the Olig2 expression inhibitor is administered in combination with dabrafenib, and thus it is expected that the Olig2 expression inhibitor of the present invention can be usefully utilized in the future to enhance the preventive and therapeutic effect of malignant melanoma.

However, the effects of the present invention are not limited to the above effects, and it should be understood that they include all effects that can be inferred from the composition of the invention described in the detailed description or claims of the present invention.

Figure 1 shows the results of confirming the expression level of Olig2 protein in proportion to the concentration of Olig2 gRNA of the CRISPR-Cas9 technique utilized to suppress Olig2 expression in 501mel cells, a malignant melanoma cell line.
Figure 2 shows the results of confirming the survival rate of melanoma cells in which the expression of Olig2 was suppressed. Figure 2a shows the survival rate of A375 cells, Figure 2b shows the survival rate of 501mel cells, Figure 2c shows the survival rate of A375 cells, and Figure 2d shows the survival rate of 501mel cells.
Figure 3a shows the results of confirming the metastatic potential of melanoma cells in which the expression of Olig2 was suppressed. Figure 3a shows the metastatic potential of A375 cells, Figure 3b shows the metastatic potential of 501mel cells, Figure 3c shows the invasive potential of A375 cells, and Figure 3d shows the invasive potential of 501mel cells.
Figure 4 shows the results of examining cell viability when CRISPR-Cas9 gRNA and dabrafenib were individually or in combination to suppress Olig2 expression in 501mel cells.

The present inventors, as a result of their research efforts to discover a substance for treating melanoma, have confirmed that when an inhibitor of the expression or activity of the transcription factor Olig2 (Oligodendrocyte transcription factor 2) and the targeted therapeutic agent dabrafenib are used in combination, the survival rate of malignant melanoma cells is significantly suppressed compared to when dabrafenib is treated alone, thereby enhancing the melanoma treatment efficacy of dabrafenib, thereby completing the present invention.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating melanoma, comprising a BRAF inhibitor and an inhibitor of expression or activity of the Olig2 (Oligodendrocyte transcription factor 2) gene or protein.

In the present invention, the term "BRAF inhibitor" may be dabrafenib, a targeted therapeutic agent used for the treatment of terminal melanoma, and in the present invention, dabrafenib is utilized for the treatment of melanoma and metastatic non-small cell lung cancer by inhibiting the expression of BRAF, a gene encoding B-Raf protein known to regulate cell growth.

In another aspect of the present invention, the present invention provides a pharmaceutical composition for enhancing the effect of preventing or treating melanoma when used in combination with a BRAF inhibitor, characterized in that the composition comprises an inhibitor of the expression or activity of transcription factor Olig2 (Oligodendrocyte transcription factor 2) as an active ingredient.

In the present invention, the term "protein" may be used interchangeably with "polypeptide" or "polypeptide" and refers to a polymer of amino acid residues. Protein applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymers.

The above-mentioned transcription factor Olig2 (Oligodendrocyte transcription factor 2, hereinafter referred to as Olig2) gene or protein expression or activity inhibitor refers to a substance that inhibits or interferes with the function of Olig2. Such inhibition or interference can be achieved by interfering with the transcription of the Olig2 gene, or inhibiting the translation of mRNA of the Olig2 gene. Alternatively, the activity of the Olig2 protein can be reduced or inactivated by specifically binding to the active site of the Olig2 protein or causing a protein structural modification.

In the present invention, the term "expression inhibitor of transcription factor Olig2 (Oligodendrocyte transcription factor 2) gene or protein" is not limited thereto, but may be any one selected from the group consisting of an antisense oligonucleotide specific for mRNA of transcription factor Olig2 (Oligodendrocyte transcription factor 2), small interfering RNA (siRNA), short hairpin RNA (shRNA), and ribozyme.

The inhibitor of the above activity may mean a substance that causes the same type of activity to be lower than the intrinsic activity that is not possessed or possessed by a genetically unmanipulated parent cell (e.g., wild type). The term "inhibitor of the activity of transcription factor Olig2 (Oligodendrocyte transcription factor 2) protein" of the present invention may be, but is not limited to, any one selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to the transcription factor Olig2 (Oligodendrocyte transcription factor 2) protein.

The above nucleotides are the building blocks of oligonucleotides and polynucleotides, and for the purposes of the present invention include both naturally occurring and non-naturally occurring nucleotides. In nature, nucleotides, such as DNA and RNA nucleotides, comprise a ribose sugar moiety, a nucleobase moiety, and one or more phosphate groups (which are absent in nucleosides). Nucleosides and nucleotides may also be referred to interchangeably as "units" or "monomers".

The meaning of "complementarily binding" above is in reference to Watson-Crick base pairing of nucleosides/nucleotides. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)-thymine (T)/uracil (U). The oligonucleotides may comprise nucleosides having modified nucleobases. For example, 5-methyl cytosine is often used in place of cytosine. The term "complementarily binding" may encompass Watson-Crick base-pairing between unmodified nucleobases and modified nucleobases (see, e.g., Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1).

The above antisense nucleotide is defined as an oligonucleotide capable of modulating the expression of a target gene by hybridizing to a target nucleic acid, particularly to an adjacent sequence on the target nucleic acid. The antisense oligonucleotide is not inherently double-stranded and is therefore not siRNA or shRNA.

The above small interfering RNA refers to RNA that can induce RNAi (RNA interference) that suppresses the activity of a gene. The above small interfering RNA can be miRNA, siRNA, etc. that can suppress the expression of the Olig2 gene. The above small interfering RNA can be of any form as long as it suppresses the expression or activity of the Olig2 gene. For example, siRNA obtained by chemical synthesis, biochemical synthesis, or in vivo synthesis, or a duplex of about 40 bases or more.

Double-stranded RNA of 10 or more bases that has been broken down in the body can be used.

The above "aptamer" refers to a nucleic acid molecule having binding activity to a predetermined target molecule. The aptamer may be RNA, DNA, a modified nucleic acid, or a mixture thereof, and may be linear or cyclic. In general, the shorter the sequence of nucleotides constituting the aptamer, the easier it is to chemically synthesize and mass-produce, the better the cost-effectiveness, the easier it is to chemically modify, the better the stability in vivo, and the lower the toxicity.

The above "antibody" means a protein molecule capable of specifically binding to an antigenic site of a protein or peptide molecule, and such an antibody can be produced by cloning each gene into an expression vector according to a conventional method to obtain a protein encoded by the marker gene, and then producing the obtained protein by a conventional method. The form of the antibody is not particularly limited, and a part of a polyclonal antibody, a monoclonal antibody, or an antibody having antigen binding property is also included in the antibody of the present invention, and not only all immunoglobulin antibodies can be included, but also special antibodies such as humanized antibodies can be included. In addition, the antibody includes not only a complete form having two full-length light chains and two full-length heavy chains, but also a functional fragment of an antibody molecule. A functional fragment of an antibody molecule means a fragment having at least an antigen binding function, and can be Fab, F(ab'), F(ab')2, and Fv, etc.

In the present invention, the term "combination" means combined administration, and is not limited thereto, but may mean concurrent (simultaneous), separate (separate) or sequential combined administration of the Olig2 inhibitor of the present invention and dabrafenib.

The present inventors confirmed through specific examples that when dabrafenib is administered in combination with an inhibitor of the expression or activity of the oilg2 gene or protein, the preventive or therapeutic effect on melanoma is improved.

In one embodiment of the present invention, when the expression of Olig2 was suppressed in melanoma cells, it was confirmed through specific experiments that the cell survival rate was significantly reduced (see Example 3-1), and that the metastatic ability and invasive ability were also significantly reduced (see Example 3-2).

In another embodiment of the present invention, when treating melanoma cells in which the expression of Olig2 was suppressed with dabrafenib, a melanoma treatment agent, it was confirmed that the growth of melanoma could be suppressed by enhancing the effect of dabrafenib (see Example 4).

Through the results described above, the inventors of the present invention specifically confirmed that when treating with an inhibitor capable of inhibiting the expression or activity of Olig2 of the present invention, the metastasis and growth of melanoma can be significantly inhibited, and when administering the inhibitor together with dabrafenib, the melanoma inhibitory effect of dabrafenib can be enhanced even at a small dose. Through these results, it was confirmed that Olig2 inhibitors can be widely utilized in the field of melanoma treatment.

The pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers in addition to the effective ingredients described above. Pharmaceutically acceptable carriers may include saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and mixtures of one or more of these components. In addition, conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.

The above pharmaceutical composition can be administered parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, transdermally or orally. The drug can be administered orally or directly to the target organ, and the dosage can be determined in light of the patient's age, sex, health status and weight, type of concurrent treatment, administration time, administration method, excretion rate, severity of the disease, frequency of treatment and nature of the desired effect. In addition, it can be administered systemically or locally, considering conditions such as the disease to be treated, necessity of site-specific treatment and amount of drug to be administered.

The pharmaceutical composition may have any pharmaceutical dosage form. For example, it may be in a form suitable for oral administration as a tablet, capsule, pill, powder, sustained-release formulation, solution, or suspension, or in a form suitable for parenteral injection as a sterile solution, suspension, or emulsion, or in a form suitable for topical administration as an ointment or cream, or in a form suitable for rectal administration as a suppository. The composition may be in the form of a tablet, pill, injection, or a combination thereof. The pharmaceutical composition may be in a unit dosage form suitable for single administration of an exact dosage.

In providing the pharmaceutical composition or performing the treatment of a disease using the pharmaceutical composition, the pharmaceutical composition may be used alone or in combination, or in combination with other therapeutic or diagnostic agents. In one specific embodiment, the pharmaceutical composition may be co-administered with other drugs typically prescribed for the intended disease according to generally accepted medical practice. In addition, the pharmaceutical composition may generally be used in vivo in a mammal, for example, a human.

Additionally, the present invention provides a method for preventing or treating melanoma, comprising administering the pharmaceutical composition to a subject.

The term "administration" as used in the present invention means providing a composition of the present invention to a subject by any suitable method.

The term "subject" as used in the present invention means a subject requiring treatment of a disease, and more specifically, a mammal such as a human or non-human primate, mouse, dog, cat, horse, and cow.

In addition, the present invention provides a use of the pharmaceutical composition for the prevention or treatment of melanoma.

Hereinafter, preferred examples are presented to help understand the present invention. However, the following examples are provided only to help understand the present invention more easily, and the content of the present invention is not limited by the following examples.

[Example]

Example 1. Experimental preparation and experimental method

1-1. Cell culture

Human melanoma cells, A735, 501mel, and HM3KO, are cultured in Dulbecco's modified eagle's medium (DMEM; Welgene Inc., Korea) supplemented with 10% fetal bovine serum (FBS) (Gibco, Los Angeles, CA, USA) and 1% penicillin/streptomycin. Human melanoma cells, MNT1, are cultured in minimum essential media (MEM; Welgene Inc., Korea) supplemented with 20% FBS, 1% penicillin/streptomycin, and 20 mM hydroxyethyl piperazineethanesulfonic acid (HEPES). Normal human melanocytes, NHEM, were cultured in medium 254 (Mediu 254, M-254-500; Invitrogen Life Technologies, Carlsbad, CA, USA) containing Human Melanocyte Growth Supplement (HMGS; Invitrogen Life Technologies, Carlsbad, CA, USA) and 1% penicillin/streptomycin at 37°C in a humidified atmosphere with 5% _CO2 .

1-2. Check cell viability

의 인큐베이터에서 추가로 1시간 동안 인큐베이션했다. 세포 생존력은 ELISA 판독기(Tecan, Mannedorf, Switzerland)에 의해 490nm 파장에서 확인하였고, 상대적 세포독성은 세포의 생존력을 백분율로 표현하여 측정한다.To determine cell viability, cells were seeded at ^1x104 cells/well in a 96-well plate, and then each cell was transfected with CRISPR-Olig2. Cell viability was determined after 24, 48, and 72 hours. To determine cell viability, an Ez-Cytox assay kit (Daeil Lab Service Co Ltd, Seoul, Korea) was used. Specifically, 10 μl of Ez-Cytox solution was added to each well-plate, and then the well-plate was incubated under 5% _CO2 conditions and 37

The cells were incubated for an additional 1 hour in a 5% CO incubator. Cell viability was determined at a wavelength of 490 nm by an ELISA reader (Tecan, Mannedorf, Switzerland), and relative cytotoxicity was measured by expressing the cell viability as a percentage.

1-3. Cell counting analysis

Cells were seeded in 96-well plates at ^1x104 cells/well and transfected with CRISPR/Cas9 plasmid. After 72 hours, the cells were treated with trypsin. 10ul of trypan blue dye (Sigma Aldrich, St. Louis, MO, USA) was mixed equally with the cell suspension. After staining the cells with trypan blue dye, the cell number was counted using a hemocytometer, repeating three times for each sample.

1-4. Real-time polymerase chain reaction analysis

For RT-PCR analysis, cells are seeded in a 6-well plate at 5 x 10 ⁵ cells/well. To extract total RNA, the melanoma cells prepared in Example 1-1 are treated with TRIZOL reagent (Takara Bio, Inc., Tokyo, Japan), and then treated with chloroform (CHCl ₃ , 99%, Junsei Chemical, Japan) at 1/5 the concentration of TRIZOL and mixed. After mixing using a vortex mixer, centrifuge at 13,000 rpm at 4°C for 15 minutes. A certain amount of the clear supernatant is taken and mixed with an equal volume of isopropanol. The tube is centrifuged at 4°C, 13,000 rpm, and inverted for 20 minutes. The supernatant was removed and the remaining pellet was diluted with diethyl pyrocarbonate treated water.

Complementary DNA (cDNA) synthesis was performed by reverse transcription polymerase chain reaction (RT-PCR) using specific primers using a SimpliAmp® thermal cycler, Thermo Fisher Scientific, MA, USA, and the specific sequences of the primers used for this are shown in Table 1 below. The synthesized cDNA was visualized using Acid Staining Solution (iNtRON Biotechnology, Seongnam, Korea) in 2% agarose gel in 1X Tris Acetate-EDTA (TAE) buffer (Elpis Biotech, Daejeon, Korea) and RedSafe® Nucleic (Bioneer Co., Daejeon, Korea).

Gene Sequence of primers
(5'→ 3`) Tm
(℃) Amp
cycles Product
size Olig2
(human) F: GAATCCGCTGGTATCCACGA 55 33 335bp R: GCGGAGCGAGACGTTTAGAA β-actin
(human) F: GGCATCGTGATGGACTCCG 59 27 610bp R: GCTGGAAGGTGGACAGCGA

1-5. Wound healing assay

Melanoma cells were seeded in 12-well plates at a density of 1 × 10 ⁵ cells/well, then transfected with the CRISPR/Cas9 plasmid. When the cells occupied approximately 90% of the plate, the monolayer was gently scratched across the center of each well with a yellow micropipette tip, and washed twice with phosphate-buffered saline (PBS). Wound healing was confirmed at the indicated times, and images were taken under a microscope. All experiments were repeated in triplicate. Differences in wound gap area were quantified compared to the control group using ImageJ software 1.45s (US National Institutes of Health, Bethesda, MD, USA). Area measurements were calculated by subtracting the area values at 24 and 48 hours from the area value at 0 hour and converting the value into a percentage of the control group.

1-6. Transwell invasion and metastasis assay

For the in vitro invasion assay, melanoma cells were seeded in 12-well plates and trypsinized 24 h after transfection of the CRISPR/Cas9 plasmid. The cells were reseeded at 5 × ₁₀₄ cells per chamber onto 8.0 μm pore size polycarbonate membranes (Corning Incorporated, Corning, NY, USA) in DMEM medium containing 1% FBS and incubated for 18 h. Specifically, the upper chamber was coated with Matrigel® (Corning, Kaiserslautern, Germany) diluted in serum-free DMEM medium, and the bottom of the upper chamber was coated with 1% gelatin. The bottom chamber was filled with DMEM medium containing 10% FBS. Cells that invaded through the Matrigel-coated membrane were fixed in 10% formalin and washed twice with PBS. To visualize the nuclei of the invaded cells, they were stained with 4',6-diamidino-2-phenylindole (DAPI). Invasive cells were detected by fluorescence microscopy at ×100 magnification. Stained nuclei were counted and quantified using ImageJ software 1.45s (US National Institutes of Health, Bethesda, MD, USA).

Example 2. Production of a cell model that suppresses Oligodendrocyte transcription factor 2 expression using CRISPR-Cas9 technology

To confirm the effect of the Olig2 inhibitor, CRISPR-Cas9 technique was used to produce cells in which the expression of the oligodendrocyte transcription factor 2 (Olig 2) was suppressed in 501mel cells, a malignant melanoma cell line. Specifically, the cells prepared in Example 1-1 were transfected with sc-400670-KO-2: Olig2 CRISPR/Cas9 KO plasmid (h2) (Santacruz Biotechnology, Dallas, TX, USA), which consists of three different gRNA plasmids encoding 20 nt guide sgRNA and Cas9 nuclease, using UltraCruz Transfection Reagent (Santacruz Biotechnology, Dallas, TX, USA) for 12 hours. The three gRNAs of the above plasmid can be gRNA1: 5`-GGCTGCGTCGTCCACCAAGA-3`, gRNA2: 5`-CACCTCGTCGTCTACGTCGT-3`, or gRNA3: 5`-CGCCCATAGCCGACACGTGG-3`.

As a result of confirming the Olig 2 expression level of 501 mel cells produced by the above method, it was confirmed that an Olig2 suppression cell model was produced in which the protein expression level of Olig2 was suppressed in proportion to the treatment concentration of Olig2 gRNA, as shown in Fig. 1.

Example 3. Confirmation of changes in melanoma cells with suppressed expression of oligodendrocyte transcription factor 2

3-1. Check survival rate

In order to confirm the cell survival rate according to the inhibition of Olig2 protein expression in the malignant melanoma cells (A375 cells and 501mel cells) in which the expression of Olig2 was suppressed as manufactured in Example 2 above, the survival rate according to the administered concentration of CRISPR-Olig2 gRNA was specifically confirmed. As a result of confirming the cell survival rate on days 1, 2, and 3 of the malignant melanoma cell model in which the protein was suppressed, it was confirmed that the cell survival rate significantly decreased, as shown in Table 2 (501mel cell data) below, Fig. 2a (A375 cells), and Fig. 2b (501mel cells). When the cell number was confirmed through cell counting analysis, it was confirmed that the cell number significantly decreased, as shown in Fig. 2c (A375 cells) and Fig. 2d (501mel cells).

Con CRISPR-Olig2 1μg CRISPR-Olig2 2μg % of Con Day1 100.00 87.45 86.63 Day2 100.00 86.14 73.90 Day3 100.00 76.66 71.02

3-2. Confirmation of metastasis

To determine whether changes in the metastatic and invasive potential of melanoma occur in cells in which Olig2 is suppressed (A375, 501mel), Examples 1-5 (wound healing assay) and 1-6 (transwell assay) were performed.

As a result of performing a wound healing assay by comparing with the control group, in the case of A375 cells, as shown in Fig. 3a, after 24 hours, the group treated with 1 μg of gRNA for Olig2 inhibition showed a 9 ± 10.0% decrease, and the group treated with 2 μg of gRNA showed a 16 ± 7.5% decrease, and after 48 hours, the group treated with 1 μg of gRNA showed a 51 ± 3.8% decrease, and the group treated with 2 μg of gRNA showed a 62 ± 16.5% decrease. In the case of 501mel cells, as shown in Fig. 3b,

After 24 hours, there was an 8 ± 9.5% decrease in the group treated with 1 μg of gRNA for Olig2 inhibition, an 18 ± 17.0% decrease in the group treated with 2 μg of gRNA, and after 48 hours, there was a 28 ± 18.6% decrease in the group treated with 1 μg of gRNA, and a 55 ± 10.3% decrease in the group treated with 2 μg of gRNA.

The number of cells that migrated through the pores of the chamber was confirmed compared to the control group through a transwell migration assay. As a result, as shown in Fig. 3c, after 18 hours, it was confirmed that the group treated with 1 μg of gRNA for Olig2 inhibition had a 63 ± 5.2% decrease, and the group treated with 2 μg of gRNA had a 58 ± 5.0% decrease. In order to confirm the invasion ability of melanoma cells, matrigel was diluted with 0% FBS DMEM medium instead of the extracellular matrix of the transwell membrane chamber, and then coated on the upper chamber, and the cells that had invaded through the matrigel were stained with DAPI and confirmed compared to the control group. As a result, as shown in Fig. 3d, it was confirmed that the relative cell invasion was confirmed to be decreased by 48 ± 8.2% in the group treated with 1 μg of gRNA for Olig2 inhibition, and by 54 ± 8.6% in the group treated with 2 μg of gRNA.

Through the above results, the present inventors confirmed that when Olig2 is inhibited, the metastasis of melanoma can be significantly inhibited.

Example 4. Confirmation of the survival rate of malignant melanoma cells when Dabrafenib is treated in cells with suppressed expression of oligodendrocyte transcription factor 2

In order to specifically confirm the combined administration effect of the Olig2 composition of the present invention, Dabrafenib, a melanoma treatment agent that has already been approved by the US FDA as a B-Raf inhibitor, was treated to normal cells or 501mel cells in which Olig2 expression was suppressed, and the cell survival rate was confirmed after 3 days. As a result, as shown in Fig. 4 and Table 3 below, it was confirmed that when Dabrafenib was administered to malignant melanoma cells in which Olig2 expression was suppressed, the growth inhibition effect of melanoma cells was more excellent than when Dabrafenib was treated to melanoma cells in which Olig2 was expressed.

Con CRISPR-Olig2 Dabrafenib CRISPR-Olig2
+ Dabrafenib % of Con 100.00 72.29 60.89 49.34

Through the above results, it was confirmed that when the Olig2 inhibitor of the present invention is administered in combination with dabrafenib, which is currently used as a treatment for melanoma, the growth of melanoma can be inhibited more effectively than when dabrafenib is administered alone.

The above description of the present invention is for illustrative purposes only, and those skilled in the art will readily appreciate that the present invention can be easily modified into other specific forms without changing the technical idea or essential characteristics of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (9)

delete delete A pharmaceutical composition for preventing or treating metastasis of melanoma, comprising dabrafenib and an inhibitor of expression of Olig2 (Oligodendrocyte transcription factor 2) gene or protein,
A pharmaceutical composition, wherein the expression inhibitor of the Olig2 (Oligodendrocyte transcription factor 2) gene or protein is any one selected from the group consisting of an antisense oligonucleotide, small interfering RNA (siRNA), short hairpin RNA (shRNA), ribozyme, and CRISPR/cas9 and gRNA specific to the transcription factor Olig2 (Oligodendrocyte transcription factor 2) mRNA.
A pharmaceutical composition for enhancing the effect of preventing or treating metastasis of melanoma in combination with dabrafenib, characterized in that the composition comprises an inhibitor of the expression of the transcription factor Olig2 (Oligodendrocyte transcription factor 2) gene or protein as an active ingredient.
A pharmaceutical composition, wherein the expression inhibitor of the Olig2 (Oligodendrocyte transcription factor 2) gene or protein is any one selected from the group consisting of an antisense oligonucleotide, small interfering RNA (siRNA), short hairpin RNA (shRNA), ribozyme, and CRISPR/cas9 and gRNA specific to the transcription factor Olig2 (Oligodendrocyte transcription factor 2) mRNA.
delete delete delete In paragraph 4,
A composition characterized in that the above combination is administered simultaneously, separately or sequentially.
delete