KR20210121767A - Use of ik associated with splicesomes - Google Patents

Abstract

The present invention relates to a use of IK, which is a spliceosome-related protein, for preventing or treating cancer. When IK of the present invention is inhibited, splicing that cuts intron 1 of ATM kinase pre-mRNA is inhibited, thereby inhibiting normal expression of ATM. Therefore, an IK inhibitor can be usefully used for the prevention or treatment of cancer induced by ATM.

Description

Use of IK, a splicesome-related protein {USE OF IK ASSOCIATED WITH SPLICESOMES}

The present invention relates to the use of IK, a splicesome-related protein, for preventing or treating cancer.

Splicesomes are large complex molecules that remove introns from pre-mRNAs. Splicesomes associated with more than 150 accessory proteins are formed from five different small nuclear ribonucleoproteins (snRNPs). In the first step of splicing, the U1 snRNP recognizes the 5'-splice site (SS) region, and the U2 snRNP binds to the U2 cofactor (U2AF) to assemble into the branch site (BS). Tri-U4/U6·U5 snRNPs assemble in U1 and U2 assemblies, and helicase Prp28 is involved in the replacement of U1 snRNPs at the 5'-splice site with U6 snRNAs. B-specific proteins, including IK and SMU1, constitute the B complex. The B complex is transformed into an activated splicesome (Bact) by unwinding of the U4/U6 RNA duplex induced by the helicase Brr2. Residual U6 snRNP interacts with U2 snRNA. During the B activation process, B-specific proteins, including IK and SMU1, are dissociated. As mentioned above, in addition to the splicing functions of IK and SMU1, IK is involved in cell cycle regulation.

Meanwhile, activation of ataxia-talangiectasia mutated (ATM) kinase is responsible for the initial process of DNA damage repair. Upon DNA damage, activated ATM results in subsequent phosphorylation of target proteins at lower levels of the signaling pathway, including CHK1, CHK2 and p53. This phosphorylation initiates the recruitment of damage repair factors in DNA lesions to repair DNA double-stand breaks (DSBs). According to previous studies, ATM and splicesomes mutually regulate. When RNA polymerase II (RNAPII) encounters a DNA lesion,

It is replaced by snRNP forming a splicesome and R-loop formation is promoted, resulting in the activation of ATM to repair damage. On the other hand, activation of ATM is known to regulate pre-mRNA splicing. However, the mechanism of ATM pre-mRNA splicing by the splicesome has not yet been elucidated.

ATM kinase also responds to direct double-strand breaks caused by ionizing radiation and common chemotherapy, such as topoisomerase-II inhibitors (doxorubicin, etoposide), but also single-stranded versus double-stranded breaks during replication. It responds to topoisomerase-I inhibitors (eg, irinotecan and topotecan) via conversion. ATM kinase inhibition can potentiate the activity of any of these agents, and consequently ATM kinase inhibitors are expected to be used in the treatment of cancer.

Accordingly, the present inventors confirmed that IK was involved in splicing of intron 1 of ATM pre-mRNA, and confirmed that it was not related to splicing of ATR. Therefore, it was confirmed that IK stabilization is very important for proper splicing of ATM, and it was confirmed that if IK was inhibited, cancer caused by ATM kinase could be prevented or treated.

An object of the present invention is to provide a composition for preventing or treating cancer comprising an IK inhibitor as an active ingredient.

Another object of the present invention is to provide a method for inhibiting the splicing of ATM protein by treating the cell with the inhibitor.

The present invention provides a pharmaceutical composition for preventing or treating cancer comprising an IK inhibitor as an active ingredient.

In addition, in the present invention, the IK is represented by SEQ ID NO: 6.

In addition, in the present invention, the IK inhibitor is an IK expression inhibitor or activity inhibitor.

In addition, in the present invention, the expression inhibitor is any one or more of the group consisting of antisense nucleotides, siRNA, shRNA, and ribozyme that are complementary to IK gene mRNA.

In addition, the present invention, the activity inhibitor is characterized in that at least one selected from the group consisting of compounds, peptides, peptidomimetics, matrix analogues, aptamers, and antibodies that bind to IK protein.

In addition, in the present invention, the composition is administered simultaneously, separately or sequentially with radiation therapy.

In addition, in the present invention, the composition comprises doxorubicin, irinotecan, topotecan, etoposide, mitomycin, bendamustine, chlorambucil, cyclophosphamide, ifosfamide, carmustine, melphalan and bleomycin. administered simultaneously, separately or sequentially with one or more additional anti-tumor agents selected from the group consisting of.

The present invention also provides that the cancer is cervical cancer, colorectal cancer, glioblastoma, gastric cancer, ovarian cancer, diffuse large B-cell lymphoma, chronic lymphomaleukemia, acute myeloid leukemia, head and neck squamous cell carcinoma, breast cancer, hepatocellular carcinoma, small cell lung cancer And it is characterized in that it is one selected from the group consisting of non-small cell lung cancer.

In addition, the present invention is characterized in that the IK is involved in the splicing of ATM (ataxia-talangiectasia mutated) kinase pre-mRNA.

In the present invention, the splicing is to cut intron 1 between exon 1 and exon 2 of ATM pre-mRNA.

In addition, the present invention is characterized in that the cancer is a cancer mediated by ATM kinase.

Also, according to the present invention, the cancer is characterized in that the expression of the IK gene is increased.

In addition, the present invention provides a method for inhibiting ATM pre-mRNA splicing by administering an IK inhibitor to a sample in vitro.

In the present invention, the splicing of the ATM pre-mRNA cuts the intron 1 site of the ATM pre-mRNA.

In addition, the present invention, administering a candidate material to a biological sample; And it provides a method of screening a cancer preventive or therapeutic agent comprising the step of confirming the expression level of IK in the biological sample.

In addition, the present invention is characterized in that the cancer is ATM-mediated cancer.

When the IK of the present invention is inhibited, splicing that cuts intron 1 of ATM kinase pre-mRNA is inhibited, thereby inhibiting the normal expression of ATM. Therefore, IK inhibitors can be usefully used for the prevention or treatment of cancer induced by ATM.

The accompanying drawings are intended to explain the contents of the present invention in more detail to those skilled in the art, but the technical spirit of the present invention is not limited thereto.
1 is a diagram confirming that IK loss of function induces abnormal fragmentation of chromosomes.
2 is a diagram showing the relationship between IK malfunction and ATM signal path.
Figure 2A is a diagram confirming the levels of IK, pATM S1981, ATM, pATR S428 and ATR after transducing the siRNA of SEQ ID NO: 1 for 47 hours and then processing for an additional hour.
Figure 2B is a diagram confirming the levels of IK, pATM S1981, ATM, pATR S428, and ATR by 24 hours after transduction of the siRNA of SEQ ID NO: 1 into cells, followed by treatment with a DNA damage inducer for an additional 24 hours.
Figure 2C is a diagram confirming the levels of IK, pATM S1981, ATM, pATR S428, ATR, and γH2AX 24 hours after transduction of the siRNA of SEQ ID NO: 1 into cells, treated with HU (hydoxyurea) for an additional 24 hours.
Figure 2D, 24 hours after transduction of the siRNA of SEQ ID NO: 1 into cells, treated with 2mM HU (hydoxyurea) for an additional 24 hours, and stained with DAPI, anti-pATM, and anti-γH2AX antibody, Figure 2E is a diagram showing the quantification of staining.
2F is a diagram illustrating the transduction of the siRNA sequence of SEQ ID NO: 1 for an additional 48 hours after transducing human or mouse IK cells into cells for 18 hours, and measuring the levels of IK and ATM.
2G, 24 hours after transduction of the siRNA of SEQ ID NO: 1 into cells, treated with 100 μM BrdU for an additional 24 hours, stained with anti-BrdU antibody, and a representative image obtained by confocal laser microscopy. (Graphically the percentage of cells exhibiting more than 15 BrdU foci (n>162) per nucleus).
Figure 2H shows that the siRNA of SEQ ID NO: 1 was transduced for 48 h, treated with 10 μM camptothecin (CPT) or etoposide (ETP) for an additional 24 h, and cell growth was further analyzed using the IncuCyte live cell imaging system. It was monitored for 24 hours.
Figure 3a is a diagram showing the level of ATM mRNA was measured after transducing cells with siRNA of SEQ ID NO: 1 for 48 hours in order to confirm the relationship between IK and ATM mRNA splicing.
Figure 3b is a diagram in which cells were transduced with the siRNA of SEQ ID NO: 1 for 48 hours, and the levels of ATM, ATR, CDC25A/B/C and E2F1 mRNA were measured.
FIG. 3c is a diagram of measuring the level of ATM mRNA spliced with intron 1 among ATM pre-mRNAs measured in FIG. 3b .
FIG. 3D is a diagram illustrating cells transduced with FLAG-IK for 18 hours and transduced with the siRNA of SEQ ID NO: 1 for an additional 48 hours, and the levels of IK and ATM mRNA were measured.
Figure 3e, cells were transduced with HA-IK for 24 hours, IK, ATM and ATR mRNA levels were measured.

The present invention relates to a composition for preventing or treating cancer comprising an IK cytokine (hereinafter referred to as "IK") inhibitor as an active ingredient.

When the IK of the present invention is inhibited, splicing that cuts intron 1 of ATM kinase pre-mRNA is inhibited, thereby inhibiting the normal expression of ATM. Therefore, IK inhibitors can be usefully used for the prevention or treatment of cancer induced by ATM.

In one embodiment of the present invention, the IK gene may be represented by SEQ ID NO: 6.

In one embodiment, the IK inhibitor may be an inhibitor of expression or activity of IK.

In one embodiment, the inhibitory agent may be an antisense nucleotide, siRNA, shRNA or ribozyme that complementarily binds to the mRNA of the IK gene.

The expression inhibitor may be an siRNA represented by SEQ ID NOs: 1 to 5.

As used herein, the term "antisense nucleotide" refers to DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to a specific mRNA sequence, and inhibits the translation of mRNA into protein by binding to a complementary sequence in mRNA. Antisense sequence refers to a DNA or RNA sequence that is complementary to IK mRNA and capable of binding to IK mRNA, and is intended for translation, translocation into the cytoplasm, maturation or any other whole biological process of IK mRNA. It may inhibit essential activity for function The length of the antisense nucleic acid is 6 to 100 bases, preferably 8 to 60 bases, and more preferably 10 to 40 bases.

The antisense nucleic acid may be modified at one or more bases, sugars or backbone positions to enhance efficacy (De Mesmaeker et al, Curr Opin Struct Biol, 5(3):343-55 (1995)) nucleic acid backbone. can be modified with phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyls, cycloalkyls, short chain heteroatomics, heterocyclic intersaccharide bonds, and the like. Antisense nucleic acids may also contain one or more substituted sugar moieties. Antisense nucleic acids may include modified bases. Modified bases include hypoxanthine, 6-methyladenine, 5-me pyrimidine (particularly 5-methylcytosine), 5-hydroxymethylcytosine (HMC), glycosyl HMC, gentobiosyl HMC, 2-aminoadenine, 2 -Thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine, 2,6-diaminopurine, etc. There is this. In addition, the antisense nucleic acid of the present invention may be chemically bound to one or more moieties or conjugates that enhance the activity and cell adsorption properties of the antisense nucleic acid. Cholesterol moiety, cholesteryl moiety, cholic acid, thioether, thiocholesterol, fatty chain, phospholipid, polyamine, polyethylene glycol chain, adamantane acetic acid, palmityl moiety, octadecylamine, hexylamino-carbonyl-oxy fat soluble moieties such as cholesterol moieties, and the like. Oligonucleotides comprising a fat-soluble moiety and methods of preparation are well known in the art (US Pat. Nos. 5,138,045, 5,218,105 and 5,459,255). The modified nucleic acids have increased stability to nucleases. and increase the binding affinity between the antisense nucleic acid and the target mRNA. Antisense oligonucleotides can be synthesized in vitro by a conventional method and administered in vivo or antisense oligonucleotides can be synthesized in vivo. One example of synthesizing antisense oligonucleotides in vitro is the use of RNA polymerase I. One example of allowing antisense RNA to be synthesized in vivo is to transcribe antisense RNA by using a vector in which the origin of the recognition site (MCS) is in the opposite direction. Such antisense RNA preferably has a translation stop codon in the sequence so that it is not translated into the peptide sequence.

The design of antisense oligonucleotides that can be used in the present invention can be easily prepared according to methods known in the art (Weiss, B (ed): Antisense Oligodeoxynucleotides and Antisense RNA: Novel Pharmacological and Therapeutic Agents, CRC Press, Boca Raton). , FL, 1997; Weiss, B, et al, Antisense RNA gene therapy for studying and modulating biological processes Cell Mol Life Sci, 55:334-358(1999)

In the present invention, the term “shRNA (small hairpin RNA or short hairpin RNA)” refers to a sequence of RNA that makes a strong hairpin turn, which can be used to silence gene expression through RNA interference. For shRNA, a vector for cell introduction is used, and the U6 promoter capable of expressing shRNA is mainly used. These vectors are always passed on to daughter cells so that gene silencing can be inherited. The shRNA hairpin structure is degraded into siRNA, an intracellular machinery, and bound to an RNA-induced silencing complex. The above-described complex binds to and degrades mRNA corresponding to the siRNA bound thereto. shRNA is transcribed by RNA polymerase III, and shRNA production in mammalian cells can also trigger an interferon response, as the cell recognizes shRNA as a viral attack and seeks a defense mechanism. In addition, shRNA can be used in plants and other systems, and the U6 promoter is not required. In the case of plants, the cauliflower mosaic virus (CaMV) 35S promoter, which is a traditional promoter with very strong continuous expression ability, can be used.

As used herein, the term “small interference RNA (siRNA)” refers to a nucleic acid molecule capable of mediating RNA interference or gene silencing (see: WO 00/44895, WO 01/36646, WO 99/32619, WO 01) /29058, WO 99/07409 and WO 00/44914).

Since siRNA can inhibit the expression of a target gene, it is provided as an efficient gene knockdown method or as a gene therapy method. siRNA was first discovered in plants, worms, fruit flies and parasites, but recently siRNA was developed and applied to mammalian cell research (Degot S, et al 2002; Degot S, et al 2004; Ballut L, et al2005). .

The siRNA molecule of the present invention may have a structure in which the sense strand and the antisense strand are positioned opposite to each other to form a double strand. In addition, the siRNA molecule of the present invention may have a single-stranded structure having self-complementary sense and antisense strands. The term complementary as used herein is meant to encompass imperfect complementarity as well as 100% complementarity, preferably 90% complementarity, more preferably 98% complementarity, and most preferably 100% complementarity. do. In this specification, when expressing 100% complementarity, it is specifically described as completely complementary. siRNA is not limited to the complete pairing of the double-stranded RNA segments that pair with each other, but is not paired by mismatch (the corresponding bases are not complementary), bulge (there is no base corresponding to one chain), etc. It may contain parts that are not. The total length is from 10 to 100 bases, preferably from 15 to 80 bases, even more preferably from 20 to 70 bases.

The siRNA is composed of a sense sequence of 15 to 30 mers selected from the nucleotide sequence of the mRNA of the gene encoding the IK protein and an antisense sequence complementary to the sense sequence, wherein the sense sequence is Although not particularly limited thereto, it is preferably composed of 25 bases.

The siRNA end structure may have either a blunt end or a cohesive end as long as it can suppress the expression of the IK gene by the RNAi effect. The adhesive end structure may have both a 3'-end protrusion structure and a 5'-end protrusion structure. The siRNA molecule of the present invention may have a form in which a short nucleotide sequence (eg, about 5-15 nt) is inserted between the self-complementary sense and antisense strands, and in this case, the siRNA molecule formed by expression of the nucleotide sequence is intramolecular A hairpin structure is formed by hybridization, and a stem-and-loop structure is formed as a whole. This stem-and-loop structure can be processed in vitro or in vivo to generate active siRNA molecules capable of mediating RNAi. According to a preferred embodiment of the present invention, the siRNA of the present invention has a sequence included in the nucleotide sequence described in SEQ ID NO: 1 .

According to a preferred embodiment of the present invention, an RNAi (RNA interference) molecule that specifically binds to an IK gene, which is an active ingredient of the composition of the present invention, may be inserted into a gene carrier.

According to a preferred embodiment of the present invention, the gene carrier of the present invention is a plasmid, a recombinant adenovirus (Thimmappaya, B et al, Cell, 31:543-551 (1982); and Riordan, JR et al, Science, 245:1066). -1073 (1989)), Adeno-associated viruses (AAV) ( LaFace et al, Viology, 162:483486 (1988), Zhou et al, Exp Hematol (NY), 21:928-933 (1993) ), Walsh et al, J Clin Invest, 94:1440-1448 (1994) and Flotte et al, Gene Therapy, 2:29-37 (1995)), retroviruses (Mann et al, Cell, 33:153-159) (1983)), lentivirus (Wang G et al, J Clin Invest 104(11):R55-62(1999)), herpes simplex virus (Chamber R, et al, Proc Natl Acad Sci USA 92:1411-1415) (1995)), Basinia virus (Puhlmann M et al, Human Gene Therapy 10:649-657 (1999)), liposomes or niosomes, most preferably recombinant lentiviruses.

In one embodiment, the activity inhibitor may be a compound, peptide, peptidomimetic, matrix analogue, aptamer or antibody that binds to an IK protein.

In one embodiment, the disease mediated by ATM kinase may be cancer. wherein said cancer is from the group consisting of colorectal cancer, glioblastoma, gastric cancer, ovarian cancer, widespread large B-cell lymphoma, chronic lymphoma leukemia, acute myeloid leukemia, head and neck squamous cell carcinoma, breast cancer, hepatocellular carcinoma, small cell lung cancer and non-small cell lung cancer. It may be one selected type, and preferably, the cancer may be cervical cancer.

In one embodiment of the present invention, IK may be characterized in that it is involved in splicing of ATM (ataxia-talangiectasia mutated) kinase pre-mRNA, wherein the splicing is exon 1 and exon 2 of ATM pre-mRNA. It may be cleaving intron 1 in between.

Accordingly, the cancer of the present invention may be a cancer in which the expression of the IK gene is increased.

In one embodiment of the present invention, the composition is administered simultaneously with, separately or sequentially with radiation therapy.

Radiation therapy in the present invention may include one or more of the following treatment regimen categories:

(i) external radiation therapy using electromagnetic radiation, and intraoperative radiation therapy using electromagnetic radiation;

(ii) internal radiation therapy or brachytherapy, including intra-tissue radiation therapy or intraluminal radiation therapy;

or

(iii) systemic radiation therapy including, but not limited to, iodine 131 and strontium 89.

In any embodiment, the radiation therapy is selected from the group consisting of one or more of the categories of radiation therapy listed in points (i)-(iii) above.

Chemotherapy in the present invention may include one or more of the following anti-tumor agent categories:

(i) anti-neoplastic agents and combinations thereof, eg, DNA alkylating agents (eg, cisplatin, oxaliplatin, carboplatin, cyclophosphamide, nitrogen mustard, such as ifosfamide, bendamustine, melphalan, chlorambucil, busulfan, temozolamide and nitrosoureas such as carmustine); antimetabolites (eg gemcitabine and anti-hydrochloric acids such as fluoropyrimidines such as 5-fluorouracil and tegafur, raltitrexed, methotrexate, cytosine arabinoside, and hydro hydroxyurea); anti-tumor antibiotics (e.g., anthracyclines, e.g., adriamycin, bleomycin, doxorubicin, liposomal doxorubicin, pyrarubicin, daunomycin, valrubicin, epirubicin, idarubicin, mitomycin- C, dactinomycin, amrubicin and mithramycin); antimitotic agents (eg, vinca alkaloids such as vincristine, vinblastine, vindesine and vinorelbine and taxoids such as taxol and taxotere and polokinase inhibitors); and topoisomerase inhibitors (eg, epipodophyllotoxins such as etoposide and teniposide, amsacrine, irinotecan, topotecan and campotecin); inhibitors of DNA repair mechanisms such as CHK kinase; DNA-dependent protein kinase inhibitors; inhibitors of poly(ADP-ribose) polymerase (PARP inhibitors, including olaparib); and Hsp90 inhibitors such as tenespimycin and retaspimycin, inhibitors of ATR kinases (eg AZD6738); and inhibitors of WEE1 kinase (eg, AZD1775/MK-1775);

(ii) antiangiogenic agents, eg, those that inhibit the effect of vascular endothelial growth factor, eg, the anti-vascular endothelial cell growth factor antibody bevacizumab and eg VEGF receptor tyrosine kinase inhibitors , for example, vandetanib (ZD6474), sorafenib, vatalanib (PTK787), sunitinib (SU11248), axitinib (AG-013736), pazopanib (GW786034) and cediranib (AZD2171); compounds as disclosed in international patent applications WO97/22596, WO 97/30035, WO 97/32856 and WO 98/13354; and compounds that act by other mechanisms (e.g., linomide, inhibitors of integrin αvβ3 function, and angiostatin), or inhibitors of angiopoietin and its receptors (Tie-1 and Tie-2), inhibitors of PLGF, inhibitors of delta-like ligands (DLL-4);

(iii) immunotherapy methods, e.g., cytokines such as interleukin 2, interleukin 4 or granulocyte macrophage colony stimulating factor, including, e.g., ex vivo and in vivo methods for increasing the immunogenicity of patient tumor cells transfection with a method for reducing T cell anergy or regulatory T-cell function; a method of enhancing a T-cell response to a tumor, e.g., CTLA4 (e.g., ipilimumab and tremalimumab), B7H1, PD-1 (e.g., BMS-936558 or AMP-514); a blocking antibody to PD-L1 (eg MEDI4736), and an agonist antibody to CD137; methods using transfected immune cells such as cytokine transfected dendritic cells; Methods using cytokine transfected tumor cell lines, antibodies to tumor-associated antigens, and antibodies lacking the target cell type (eg, a non-conjugated anti-CD20 antibody, eg, rituximab, radiation labeled anti-CD20 antibodies Bexar and zevalin, and anti-CD54 antibody Campas); methods using anti-genotypic antibodies; how to enhance natural killer cell function; and methods using antibody-toxin conjugates (eg, anti-CD33 antibody Mylotarg); immunotoxins such as moxetumumab fasodotox; agonists of Toll-like receptor 7 or Toll-like receptor 9;

(iv) efficacy enhancers, for example leucovorin.

Accordingly, in one embodiment, for use for the treatment of cancer, a composition of the present invention is administered in combination with an additional anti-tumor agent.

In one embodiment, for use in the treatment of cancer, the compositions of the present invention and cisplatin, oxaliplatin, carboplatin, valrubicin, idarubicin, doxorubicin, pyrarubicin, irinotecan, topotecan, amrubicin, epi selected from the group consisting of rubicin, etoposide, mitomycin, bendamustine, chlorambucil, cyclophosphamide, ifosfamide, carmustine, melphalan, bleomycin, olaparib, MEDI4736, AZD1775 and AZD6738 At least one additional anti-tumor agent is provided.

Another embodiment of the present invention provides a method for inhibiting ATM pre-mRNA splicing by administering an IK inhibitor to a sample in vitro.

The splicing of the ATM pre-mRNA may be cleaving the intron 1 region of the ATM pre-mRNA.

In another embodiment of the present invention, administering a candidate material to a biological sample; and

It provides a method of screening a cancer preventive or therapeutic agent comprising the step of confirming the level of IK expression in the biological sample.

The cancer may be characterized as an ATM mediated cancer.

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[Test method]

1. cell line

HeLa and HEK 293T cells were cultured in DMEM (Dulbecco's modified Eagle's medium; Hyclone) containing heat-inactivated fetal bovine serum at 37° C., in a CO ₂ incubator humidified to 5%.

Cells were treated with thymidine (Thy), mitomycin C (MMC; mitomycin C), neocarzinostatin (NCS; neocarzinostatin), camptothecin (CPT; camptothein), etoposide (ETP; etoposide) or hydroxyurea ( HU; treated with a DNA damage reagent such as hydroxyurea). In addition, cells were treated with protein synthesis inhibitor cycloheximide (CHX; cycloheximide) and proteasome inhibitor MG132.

2. IK plasmid preparation with full-length sequence

Human full-length IK cDNA was purchased from Origin (OriGene Technologies, Inc., MD, Rockville) and cloned into pcDNA 3.1, pcDNA 3.0 and pCMV-tag-2B vectors and used. The mouse full-length IK cDNA was purchased from Origene and cloned into pcDNA 3.1 vector. Plasmid transduction was performed using a polyethylenimine (PEI; polyethylenimine) solution, X-tremeGENE HP DNA transduction reagent, and jetPRIME (Polyplus) reagent according to the manufacturer's manual.

3. RNA Interference Analysis

For RNA interference analysis, IK siRNA duplex sequences were prepared.

Specifically, 5'-CAAAGGUUGCAAGAUGUUU-3' of SEQ ID NO: 1; 5'-CUACCAAGGAGUUGAUCAA-3' of SEQ ID NO: 2; 5'-GCAUUCCAGUAUGGUAUCA-3' of SEQ ID NO: 3; 5'-AGACCACACUGACCACAAA-3' of SEQ ID NO: 4; 5'-AGCUGAGAUUGCCAGCAAA-3' of SEQ ID NO: 5 was prepared. In the present invention, a concentration of 20 nM was used.

siRNA was synthesized in bioneer, and for DUB siRNA screening, the bioneer screening AccuTarget™ human ubiquitin siRNA set was used. According to the manufacturer's manual, siRNA was transduced into HeLa cells using Lipofectamine RNAiMax transduction reagent (Invitrogen).

For analysis of restoration of IK, cells were transduced with IK siRNA 18 hours after transduction with plasmids expressing human or mouse IK. Cell lysates were obtained 48 hours after transduction with IK siRNA.

4. Metaphase spreads

The cells were treated with colcemid (Sigma-Aldrich) at a concentration of 0.2 μg/mL for 90 minutes to stop the cell cycle, and then incubated in 0.075 M KCl at 37° C. for 15 minutes to harvest the cells, followed by methanol: It was fixed in an acetic acid (3:1; v/v) solution.

Metaphase spread was performed by dropping the cell suspension onto slides pretreated with _{ddH 2 O.} The slides were dried at 42° C. for 60 minutes, and then stained with DAPI loading medium (Sigma-Aldrich) in Gurr Buffer for 3 minutes. After rinsing with fresh Gurr Buffer and distilled water, the slides were completely dried and the cells were observed under a Zeiss microscope.

5. Antibodies

The primary antibody was used for immunoblotting and immunofluorescence.

Rabbit polyclonal anti-IK (Santa Cruz, sc-1335485), Rabbit polyclonal anti-IK antibody (Bethyl Laboratories, A301-708A), Rabbit monoclonal anti-pATM S1981 antibody (Cell Signaling, #5883), Rabbit monoclonal Anti-ATM antibody (Cell Signaling, #2873), rabbit polyclonal anti-pATR S428 antibody (Cell Signaling, #2853), rabbit monoclonal ATR antibody (Cell Signaling, #13934), rabbit monoclonal anti-pCHK1 S345 antibody ( Cell Signaling, #2348), rabbit polyclonal anti-pCHK1 S317 antibody (Cell Signaling, #2344), rabbit polyclonal anti-pCHK2 T68 antibody (Cell Signaling, #2661), mouse monoclonal anti-HA antibody (Santa Cruz, sc-7392), mouse monoclonal anti-FLAG antibody (Sigma, F1804), anti monoclonal anti-GFP antibody (Spanta Cruz, sc-9996), mouse monoclonal anti-BrdU antibody (Cell Signaling, #5292). The secondary antibody was purchased from Enzo life Science (HRP-conjugated goat anti-mouse or anti-rabbit IgG (Fab) antibody).

6. Immunofluorescence

HeLa cells on the coverslip were treated with 0.1% Triton X-100 in PBS for 3 minutes, and then fixed with 4% paraformaldehyde in PBS for 10 minutes. Cells were washed twice with PBS, permeabilized with 0.5% NP-40 in PBS for 5 min, and blocked with PBS-BT (3% BSA and 0.1% Triton X-100) for 30 min at room temperature. Cells on coverslips were incubated with primary and secondary antibodies diluted in PBS-BT for 1 h at room temperature. Nuclei of fixed cells were stained with Hoechst 33258 or DAPI loading medium. Images were acquired on an LSM-700 confocal laser scanning microscope (Carl Zeiss) using a 63X oil immersion objective lens and ZEN software (Nikon).

7. Immunoblot Analysis

For immunoblot analysis, cells were lysed in lysis buffer (supplemented with 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA and 1% NP-40, soluble protein and a phosphatase inhibitor mixture (Roche)). . Cell lysates were obtained by centrifugation at 20,000 g at 4° C. for 15 minutes, and the concentration of the supernatant was quantified using a Pierce BCA protein assay kit (Thermo Scientific). Total protein lysates were prepared using 5x SDS sample buffer and heating at 99° C. for 10 minutes. Proteins were separated by electrophoresis on an SDS-polyacrylamide gel and transferred to a 0.45 μm nitrocellulose membrane. Cellulose membranes were incubated overnight with an antibody containing 3% BSA in TBS-T ((150 mM NaCl, 20 mM Tris-HCl (pH 8.0) and 0.05% Tween-20) at 4 °C, followed by TBS-T for 2 h at room temperature. HRP-conjugated goat anti-mouse or anti-rabbit IgG (Fab) (Enzo Life Sciences) was treated in 5% skim milk in 5% skim milk, after which the target protein was visualized with ECL western blotting reagent and analyzed by Fusion Solo-S image analyzer (Vilber ) was analyzed.

8. Immunoprecipitation assay

Cells were lysed with lysis buffer [50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA and 1% NP-40 supplemented with a mixture of protease and phosphatase inhibitors (Roche)]. Cell lysates were obtained by centrifugation at 20,000 g at 4° C. for 15 minutes.

For immunoprecipitation of endogenous proteins, cell lysates were incubated with 3 μg of antibody overnight, and then incubated with protein G agarose beads (Amicogen, 2010005) at 4° C. for 2 hours. Then, the immune complexes were washed 4 times with lysis buffer, the immune complexes were separated by SDS-polyacrylamide gel, and immunoblotting analysis was performed.

9. RNA Immunoprecipitation

To perform RNA immunoprecipitation, HeLa cells were resuspended in nuclear isolation buffer (1.28 M sucrose, 40 mM Tris-HCl pH 7.5, 20 mM MgCl ₂ , 4% Triton X-100) and nucleated using RIP buffer. Proteins were extracted. Anti-IK antibody was mixed with HeLa nuclear extract in RIP buffer and then incubated at 4°C overnight. After this, 30 μL of protein G-agarose beads were added and the samples were further incubated at room temperature for 3 hours. The beads were then washed 5 times with RIP buffer and RNA was extracted with TRIzol and reverse transcribed. The resulting cDNA was used as a template for performing PCR. The primers used for ATM detection were designed to demonstrate that they were due to RNA binding specifically to IK.

10. ATM mini gene splicing assay

A 262 bp human ATM mini-gene construct was cloned into the pEGFP-N2 vector. ATM fragment comprising 72 bp exon 1 with a start codon, 79-bp intron 1 (5'-GU-A-AG-3' with splicing recognition site) and 111-bp exon 2 EcoRI and BamHI restriction enzyme sites were located on both sides, and the ATM was amplified from cDNA extracted from cells with loss of IK function. The mini gene construct (0.5 μg) was transduced with 2 μL of HP DNA transduction reagent (Roche) 24 hours after siRNA transduction.

Additionally, after 24 h incubation, protein was extracted using lysis buffer, immunoblotted with anti-GFP antibody, and expression of green fluorescent protein (GFP) was observed by fluorescence microscopy.

[Example 1] IK function loss (depletion) and the relationship between abnormal fragmentation of chromosomes (fragmentation) confirmation

In order to confirm the relationship between the loss of IK function and the abnormal chromosomal structure formation, 5 different siRNAs targeting various regions of IK, including the coding region and 3'-UTR, were used (FIG. 1A; see SEQ ID NOs: 1 to 5) .

More specifically, cells transduced with 5 different siIKs were stained with the transfection Hoechst 33258 for 48 h. Changes in nuclear morphology were investigated by confocal laser microscopy, and IK levels were assessed.

As a result of the evaluation, when five IK siRNAs were introduced into cells, it was confirmed that IK expression was successfully reduced and chromosomal abnormalities were induced ( FIG. 1B ).

In addition, metaphase spread was performed using cells transduced with SEQ ID NO: 1 or SEQ ID NO: 3 siRNA. Representative images of metaphase spread were confirmed from cells with loss of IK function obtained by confocal laser microscopy.

From these results, it was confirmed that loss of IK function (knockdown) induces various atypical chromosomes ( FIG. 1C ). Thus, it was confirmed that IK plays a critical role in genomic stability and cell viability in human cells.

[Embodiment 2] Confirmation of relationship between IK and ATM signal path

In order to confirm the relationship between IK and the DNA damage repair system under cellular stress, cells in which IK function was lost were treated with NCS (neocarzinostatin), which induces DNA damage.

As a result of the treatment, it was confirmed that phosphorylation of CHK2 was slightly decreased, whereas phosphorylation of ATM and CHK1 was significantly decreased compared to phosphorylation of CHK2 (FIG. 2A).

In addition, HeLa cells with loss of IK function were treated with various DNA damage derivatives including Thy, MMC, CPT or ETP. It was confirmed that phosphorylation was increased in S1981 of ATM in cells with loss of IK function. However, it was confirmed that the ATR protein and phosphorylation levels were not affected ( FIG. 2B ).

In addition, under the DNA replication stress induced by HU treatment, a weak increase in ATM phosphorylation was confirmed in cells with a loss of IK function ( FIG. 2C ). Upon HU treatment, γH2AX lesions present along with phosphorylated ATM were identified at a low rate ( FIGS. 2D and 2E ).

In order to exclude the off-target effect of IK siRNA of the above effects, we transduced full-length human or mouse IK into IK-deficient cells.

It was confirmed that the ATM protein level was restored in IK-knockdown cells ( FIG. 2F ).

In addition, when IK function is lost, in order to confirm the effect on the ATM-mediated DNA damage repair system, the frequency of ssDNA cleavage was confirmed by performing BrdU incorporation assays.

As a result, it was confirmed that cells in which IK function was lost rapidly increased the formation of BrdU lesions, impairing the DNA repair process ( FIG. 2G ).

In addition, cells with loss of IK function were treated with CPT and ETP. It was confirmed that the number of cells was significantly reduced by the drug inducing DNA damage (FIG. 2H). Collectively, we confirmed that IK is associated with the regulation of endogenous ATM protein levels.

[Example 3] Confirmation of splicing relationship for ATM pre-mRNA intron 1 of IK

In order to confirm the relationship between the loss of IK function and splicing of ATM mRNA, the level of mRNA was checked using exon-exon-specific primers designed based on different ATM exon regions.

As a result, it was confirmed that ATM mRNA levels were decreased in cells in which IK function was lost ( FIG. 3A ).

Primers targeting the exon 1 and exon 2 regions of ATM pre-mRNA were designed and RT-PCR was performed. It was confirmed that splicing between exon 1 and exon 2 in ATM pre-mRNA was significantly reduced in cells in which IK function was lost ( FIG. 3B ).

However, it was confirmed that ATR splicing proceeded normally.

In addition, it was confirmed that splicing proceeds normally in CDC25A, CDC25B and CDC25C, which are inhibited by ATM-mediated pathway, and the E2F1 transcription factor that increases ATM pre-mRNA levels (FIG. 3B).

By measuring the level of ATM mRNA spliced with intron 1 among ATM pre-mRNAs measured in FIG. 3B, it was confirmed numerically that ATM mRNA spliced normally was reduced in cells in which IK function was lost (FIG. 3C) .

From the above results, it was confirmed that IK is an important splicing factor involved in the processing of ATM pre-mRNA. In addition, when the expression of IK was restored using the siIK-resistant expression plasmid, it was confirmed that the ATM pre-mRNA, which had been normally spliced, was increased ( FIG. 3D ).

In the case of IK overexpression, it was confirmed that there was no effect on ATM splicing (Fig. 3E).

From these results, it was confirmed that the loss of IK function caused a decrease in the splicing of exon 1 and exon 2 of ATM pre-mRNA.

The above description of the present application is for illustration, and those of ordinary skill in the art to which the present application pertains will understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present application. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. For example, each component described as a single type may be implemented in a dispersed form, and likewise components described as distributed may be implemented in a combined form.

The scope of the present application is indicated by the following claims rather than the above detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present application.

<110> SOOKMYUNG WOMEN'S UNIVERSITY Research & Business Development Foundation <120> USE OF IK ASSOCIATED WITH SPLICESOMES <130> DP200149 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> A kind of IK siRNA <400> 1 caaagguugc aagauguuu 19 <210> 2 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> A kind of IK siRNA <400> 2 cuaccaagga guugaucaa 19 <210> 3 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> A kind of IK siRNA <400> 3 gcauuccagu augguauca 19 <210> 4 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> A kind of IK siRNA <400> 4 agaccacacu gaccacaaa 19 <210> 5 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> A kind of IK siRNA <400> 5 agcugagauu gccagcaaa 19 <210> 6 <211> 1674 <212> RNA <213> Artificial Sequence <220> <223> IK mRNA <400> 6 atgccggagc gagatagtga gccgttctcc aaccctttgg cccccgatgg ccacgatgtg 60 gatgatcctc actccttcca ccaatcaaaa ctcaccaatg aagacttcag gaaacttctc 120 atgaccccca gggctgcacc tacctctgca ccaccttcta agtcacgtca ccatgagatg 180 ccaagggagt acaatgagga tgaagaccca gctgcacgaa ggaggaaaaa gaaaagttat 240 tatgccaagc tacgccaaca agaaattgag agagagagag agctagcaga gaagtaccgg 300 gatcgtgcca aggaacggag agatggagtg aacaaagatt atgaagaaac cgagcttatc 360 agcaccacag ctaactatag ggctgttggc cccactgctg aggcggacaa atcagctgca 420 gagaagagaa gacagttgat ccaggagtcc aaattcttgg gtggtgacat ggaacacacc 480 catttggtga aaggcttgga ttttgctctg cttcaaaagg tacgagctga gattgccagc 540 aaagagaaag aggaagagga actgatggaa aagccccaga aagaaaccaa gaaagatgag 600 gatcctgaaa ataaaattga atttaaaaca cgtctgggcc gcaatgttta ccgaatgctt 660 tttaagagca aagcatatga gcggaatgag ttgttcctgc cgggccgcat gacctatgtg 720 gtagacctgg atgatgagta tgctgacaca gatatcccca ccactcttat ccgcagcaag 780 gctgattgcc ccaccatgga ggcccagacc acactgacca caaatgacat tgtcattagc 840 aagctgaccc agatcctttc atacctgagg cagggaaccc gtaacaagaa gcttaagaag 900 aaggataaag ggaagctgga agagaagaaa cctcctgagg ctgacatgaa tatttttgaa 960 gacattgggg attacgtacc ctccacaacc aagacacctc gggacaagga gcgggagaga 1020 tatcgggaac gggagcgtga tcgggaaaga gacagagacc gtgaccgaga gcgagagcga 1080 gaacgagatc gggaacgaga gcgagagcgg gaccgagaga gagaagagga aaagaagaga 1140 cacagctact ttgagaagcc aaaagtagat gatgagccca tggacgttga caaaggacct 1200 gggtctacca aggagttgat caagtccatc aatgaaaagt ttgctgggtc tgctggctgg 1260 gaaggcacag aatcgctgaa gaagccagaa gacaaaaagc agctgggaga tttctttggc 1320 atgtccaaca gttatgcaga gtgctaccca gccacgatgg atgacatggc tgtggatagt 1380 gatgaggagg tggattatag caaaatggac cagggtaaca agaaggggcc cttaggccgt 1440 tgggactttg atacccagga agaatacagc gagtatatga acaacaaaga agctttgccc 1500 aaggctgcat tccagtatgg tatcaaaatg tctgaagggc ggaaaaccag gcgcttcaag 1560 gaaaccaatg acaaagcaga gcttgatcgc cagtggaaga agattagtgc aatcattgag 1620 aagaggaaga agatggaagc tgatggggtt gaagtcaaaa gaccaaaata ctaa 1674

Claims (16)

A pharmaceutical composition for preventing or treating cancer comprising an IK (IK cytokine) inhibitor as an active ingredient.
The composition of claim 1, wherein the IK is represented by SEQ ID NO: 6.
The composition of claim 1, wherein the IK inhibitor is an IK expression inhibitor or activity inhibitor.
The composition of claim 3, wherein the expression inhibitory agent is at least one antisense nucleotide, siRNA, shRNA, and ribozyme that complementarily binds to the mRNA of the IK gene.
The composition of claim 3, wherein the activity inhibitor is at least one selected from the group consisting of a compound that binds to an IK protein, a peptide, a peptidomimetic, a matrix analog, an aptamer, and an antibody.
The composition of claim 1 , wherein the composition is administered concurrently, separately or sequentially with radiation therapy.
According to claim 1, wherein the composition is doxorubicin, irinotecan, topotecan, etoposide, mitomycin, bendamustine, chlorambucil, cyclophosphamide, ifosfamide, carmustine, melphalan and bleomycin The composition is administered simultaneously, separately or sequentially with one or more additional anti-tumor agents selected from the group consisting of.
The method of claim 1 , wherein the cancer is cervical cancer, colorectal cancer, glioblastoma, gastric cancer, ovarian cancer, diffuse large B-cell lymphoma, chronic lymphomaleukemia, acute myeloid leukemia, cervical squamous cell carcinoma, breast cancer, hepatocellular carcinoma, small cell The composition, characterized in that it is one selected from the group consisting of lung cancer and non-small cell lung cancer.
The composition for preventing or treating cancer according to claim 1, wherein the IK is involved in splicing of ATM (ataxia-talangiectasia mutated) kinase pre-mRNA.
10. The composition of claim 9, wherein the splicing cleaves intron 1 between exon 1 and exon 2 of ATM pre-mRNA.
The composition of claim 1 , wherein the cancer is a cancer mediated by ATM kinase.
The composition of claim 1, wherein the cancer is a cancer in which the expression of the IK gene is increased.
A method of inhibiting splicing of ATM pre-mRNA by administering an IK inhibitor to a sample in vitro.
14. The method of claim 13, wherein the splicing of the ATM pre-mRNA cleaves the intron 1 site of the ATM pre-mRNA.
administering a candidate substance to the biological sample; and
A method of screening a cancer preventive or therapeutic agent comprising the step of confirming the level of IK expression in the biological sample.
16. The method of claim 15, wherein the cancer is an ATM mediated cancer.

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