KR20200057228A - Method of screening agents for treating cancer by targeting JAK2-STAT3-KDM3A signaling pathway - Google Patents

Abstract

The present invention provides a screening method for a cancer therapeutic agent targeting a JAK2-STAT3-KDM3A signaling pathway. The method according to the present invention can control a JAK2-STAT3 signaling pathway which is closely related to development and progression of malignant tumors through inhibition of KDM3A phosphorylation, thereby enabling the development of an effective new cancer therapeutic agent.

Description

Method of screening agents for treating cancer by targeting JAK2-STAT3-KDM3A signaling pathway}

It is related to the development of cancer therapy based on molecular mechanisms that act on the JAK2-STAT3 specific signaling pathway.

JAK2 (Janus tyrosine kinase 2) -signal transducer and activator of transcription 3 (STAT3) signaling pathways are essential pathways for cell development, cell differentiation, and homeostasis regulation. It is known to be related. Specifically, JAK2 is a non-receptor tyrosine phosphorylase that induces the cytoplasmic signal transduction process, and JAK2 is also present in the nucleus of human hematopoietic cells and is known to act as a direct phosphorylase of histone H3Y41. When JAK2 is activated, it activates transcription factor STAT3. STAT3 is phosphorylated by JAK2 and forms a stable homodimer or heterodimer to induce local activation of the target gene. This abnormal response of the JAK2-STAT3 signaling pathway has been shown to induce abnormal cell proliferation, migration and anti-cell death in various types of cancer (Griffiths DS , et al. (2011) LIF-independent JAK signaling to chromatin in embryonic stem cells uncovered from an adult stem cell disease.Nature cell biology 13 (1): 13-21 .; Marotta LL , et al. (2011) The JAK2 / STAT3 signaling pathway is required for growth of CD44 (+) CD24 (- ) stem cell-like breast cancer cells in human tumors.The Journal of clinical investigation 121 (7): 2723-2735 .; Yu H, Kortylewski M, & Pardoll D (2007) Crosstalk between cancer and immune cells: role of STAT3 in the tumour microenvironment.Nature reviews.Immunology 7 (1): 41-51.).

Therefore, suppression of the JAK2-STAT3 signal transduction axis may be a target for cancer treatment, but the development of a more effective therapeutic agent requires the identification of sub-regulators that specifically act in the signal transduction pathway and their mechanisms. KDM3A is known to activate androgen receptor target genes such as PSA and NKX3.1 by regulating the process of transcription activation in metabolism and sperm formation, and Korea Patent Publication No. 2017-0112755 uses the KDM3A gene as a marker for diagnosing blood cancer. It starts.

However, the relationship between KDM3A and JAK2-STAT3 signaling is unknown. Furthermore, no regulatory mechanism for KDM3A transcriptional activation and inhibition related to the JAK2-STAT3 signaling pathway during cancer development or progression has been identified.

This application aims to identify essential epigenetic factors necessary for the development of therapeutic agents for cancer related to activation of the JAK2-STAT3 signaling pathway.

In one aspect, the present application discloses a method for screening a substance for treating cancer associated with an abnormality in the signal transducer and activator of transcription 3 (JAK2) signaling pathway via lysine-specific demethylase 3A (KDM3A) -STAT3. .

In one embodiment, the method is to inhibit phosphorylation of the JAK2 protein and the KDM3A protein comprising the 1101 th tyrosine residue based on the sequence of SEQ ID NO: 1 that can be phosphorylated by the JAK2 protein. A first step of culturing in the presence of the expected test substance; A second step of measuring the phosphorylation degree of the tyrosine residue of the KDM3A protein; And a third step of selecting phosphorylation of the KDM3A protein when treated with a test substance, as a candidate substance capable of inhibiting the growth of cancer cells, when compared to a control group not treated with a test substance as a result of the measurement. do.

In another embodiment, the method further comprises measuring the activity of the KDM3A protein as a cofactor for the STAT2 protein in the first step, wherein the first step further comprises a STAT3 protein, and the 3 In the step, as compared to a control group not treated with a test substance as a result of the measurement, when treated with a test substance, the activity of the KDM3A protein as a cofactor for the STAT3 protein, when the transcriptional activity of the STAT3 protein is reduced, It further comprises selecting as a candidate substance for inhibiting the growth of cancer cells.

In another embodiment, in all of the above methods, the first step further comprises a histone 3 protein (H3K9, Histone 3 Lysine 9) methylated with a ninth lysine residue based on SEQ ID NO: 4, in the second step. In addition, further comprising measuring the demethylation activity of the KDM3A protein for H3K9, and compared to the control group not treated with the test result in the measurement step in step 3, when the test material is treated with the H3K9 deproteinization When methylation is reduced, it further comprises selecting it as a candidate for inhibiting the growth of cancer cells.

In another embodiment, in all of the above methods, the first and second steps are performed in eukaryotic cells expressing the respective proteins, and the cells are further added as a substance capable of activating the JAK2-STAT signaling pathway. Is processed.

In another embodiment, in all of the above methods, the first and second steps are performed on eukaryotic cells expressing the respective proteins, in particular mammalian eukaryotic cells, particularly cancer-derived mammalian eukaryotic cells, such as Hela cells, wherein the cells are JAK2. -A substance capable of activating the STAT signaling pathway, and gp (glycoprotein) 130 including IL (Interleukin) -6, IL-11, or Oncostatin M (OSM), may be activated as a mediating signal transduction substance.

In one embodiment, the material screened according to the method according to the present application can be developed and used as a treatment for solid cancer, including solid cancer, including cervical cancer, ovarian cancer, breast cancer, kidney cancer, and stomach cancer.

In another aspect, the present application also provides an antibody or antigen-binding fragment that specifically recognizes KDM3A phosphorylated with the 1101 th tyrosine residue based on the sequence of SEQ ID NO: 1. For the first time, an antibody capable of specifically recognizing KDM3A phosphorylated at residue 1101, a sub-regulator that specifically functions in the JAK2-STAT signaling pathway, was developed.

In another embodiment, the KDM3A protein 1101 th tyrosine residue is phosphorylated KMpYNAYGLI based on the sequence of SEQ ID NO: 1 (a short code representing amino acid residues, lysine, methionine, phosphorylated tyrosine, asparagine, alanine, tyrosine, glycine, respectively) , Represents leucine and isoleucine) Antibodies specifically recognizing epitopes or antigen-binding fragments thereof are provided.

In one embodiment, the antibody may be a polyclonal or monoclonal antibody.

Provided herein are methods of screening for cancer therapeutics targeting the JAK2-STAT3- KDM3A signal transduction pathway. JAK2-STAT3 is essential for regulating cell development, differentiation and homeostasis. Thus, dysregulation of the JAK2-STAT3 signaling system has been linked to the formation of malignant tumors in humans. In the present invention, it was confirmed that KDM3A serves as an essential epigenetic factor for activation of the JAK2-STAT3 signaling pathway. Specifically, it was found that KDM3A is phosphorylated by JAK2 in the nucleus, which functions as a co-activation factor in the STAT3-dependent transcription process. It was found that the JAK2-KDM3A signaling system induced by IL-6 functionally and physically links between activation of the JAK2-STAT3 signaling pathway and epigenetic control, thereby inducing a change in histone H3K9 methylation as an epigenetic genetic phenomenon. Did. This shows that inhibition of KDM3A phosphorylation can be a powerful therapeutic strategy to control the carcinogenic effects of the JAK2-STAT3 signaling pathway.

Figure 1 JAK2 phosphorylates KDM3A at the residue of tyrosine 1101 (A) Identification of the tyrosine phosphorylation site on KDM3A by LC-MS / MS. (B) Schematic diagram of KDM3A showing zinc finger (ZF) and Jumanji (JmjC) domains. The sequence around the tyrosine residue (Y, shown in red) is conserved between Drosophila (d), rat (m) and human (h). (C) The protein extract obtained from HeLa cells was pulled down with a tyrosine phosphorylated antibody, and phosphorylation of KDM3A was confirmed by immunoblot with a KDM3A antibody. (D) Operation of KDM3A (KDM3A-Y1101p) phosphorylated antibody was confirmed by dot blotting of unmodified KDM3A and phosphorylated KDM3A peptide. (E) HeLa cells knock down KDM3A using shRNA and transfect HA-KDM3A WT (WT ^R ) or HA-KDM3A Y1101A mutant (YA ^R ) resistant to shRNA. KDM3A was immunoprecipitated using HA antibody and analyzed by immunoblot with KDM3A phosphorylated antibody. (F and G) Immunoprecipitation (Co-IP) analysis of JAK1 (F) or JAK2 (G) and KDM3A in HeLa cells was performed with or without IL-6 treatment (50 ng / ml) for 2 hours. (H) Co-IP analysis of KDM3A phosphorylated with JAK1 or JAK2 was performed on HeLa cells treated with IL-6 for 2 hours. The degree of phosphorylation of KDM3A was confirmed by immunoblot analysis using KDM3A phosphorylated antibody. (I) After knock-down of JAK2 using shRNA, the degree of phosphorylation of KDM3A was confirmed by immunoblot analysis using KDM3A phosphorylated antibody in HeLa cells with or without IL-6 treatment for 2 hours. (J) Transfecting wild type (WT) of JAK2, continuously activated mutant (CA), or functionally inactive mutant (DN) into HeLa cells. Cell lysates were immunoprecipitated with KDM3A antibody, followed by immunoblot analysis with KDM3A phosphorylated antibody to confirm phosphorylated KDM3A. (K) In vitro phosphorylation analysis using JAK2 CA or DN mutants using recombinant KDM3A protein. (L-0) HeLa cells were treated with IL-6 (50 ng / ml) for the time indicated, and the phosphorylation level of KDM3A was inhibited with the JAK2 activity inhibitor AG490 (10 μM) (L) or TG101348 (2.5 μM) (M). It was confirmed by immunoblotting with KDM3A phosphorylated antibody according to the presence or absence of treatment with the Src family inhibitor PP2 (10 μM) (N) or the Abl inhibitor Asciminib (250 nM) (O).
Figure 2, KDM3A phosphorylation by JAK2 increases the demethylase activity of KDM3A (A) Analyzes the degree of H3K9me2 in HeLa and HEK293T cells after IL-6 treatment. (B) JAK2 was knocked down with shRNA, and the degree of H3K9me2 according to the presence or absence of IL-6 treatment was confirmed by immunoblot. (C) A representative image of JAK2 knock-down with shRNA, and the degree of H3K9me2 depending on the presence or absence of IL-6 treatment was confirmed by immunostaining. Cells were stained with H3K9me2 or JAK2 antibody. Nuclei are marked with DAPI. Scale bar, 10 μm. (D) Demethylation of H3K9me2 was induced by JAK2, and knock-down of KDM3A confirmed that the demethylation of JAK2-dependent H3K9me2 was broken when IL-6 was treated. (E) HeLa cells are knocked down by KDM3A shRNA and transfected with shRNA resistant forms of KDM3A WT (WT ^R ) or Y1101A (YA ^R ). Transfected cells were stained with H3K9me2 and Flag antibodies. Cell nuclei stained with DAPI. Arrows indicate the level of cells expressing KDM3A and H3K9me2. Scale bar, 10 μm. (F) Demethylation of H3K9me2 induced by IL-6 depends on KDM3A. The protein extract of HeLa cells was knocked down with KDM3A shRNA, and KDM3A WT ^R or YA ^R was transfected to confirm the degree of H3K9me2 according to the presence or absence of IL-6 treatment by immunoblot. (G and H) Oncostatin M (OSM) (20 ng / ml) (G) or IL-11 (5 ng / ml) (H), excluding IL-6 sharing gp130 mediated signaling molecules in cells After treatment with other cytokines, the level of H3K9me2 was confirmed in HeLa cells.
3 shows that KDM3A functions as a transcriptional coactivator of STAT3 in the JAK-STAT signaling pathway (A) The interaction between STAT3 and KDM3A with or without IL-6 treatment is confirmed by coimmunoprecipitation in HeLa cells. (B) ChIP analysis was performed on the MYC promoter using ILK treatment in HeLa cells, followed by JAK2, STAT3 phosphorylation, KDM3A phosphorylation and H3K9me2 antibodies. ** P <0.01, *** P <0.001 (Student's t -test). (C) Quantitative RT-PCR analysis of MYC mRNA expression after knock-down of KDM3A with shRNA after IL-6 treatment in HeLa cells. *** P <0.001 (Student's t -test). (D) Immunoblot analysis of MYC protein after knock-down of KDM3A with shRNA after IL-6 treatment in HeLa cells. (E) After treatment of IL-6 in HeLa cells, knockdown of KDM3A with shRNA and transfection of resistant KDM3A WT ^R or YA ^R confirmed MYC mRNA levels by quantitative RT-PCR. * P <0.05 (Student's t -test). (F) After treatment of IL-6 in HeLa cells, knockdown of KDM3A with shRNA and transfection of resistant KDM3A WT ^R or YA ^R confirmed the MYC protein level by immunoblot. (GI) Transfected as indicated on HEK293T cells with M67 promoter-luciferase reporter plasmid in response to STAT. Luciferase reporter activity was measured 48 hours after transfection and normalized by β-galactosidase activity. Three independent experiments. * P <0.05, ** P <0.01 (Student's t -test).
Figure 4 is a photomicrograph of KDM3A phosphorylation increases cell proliferation and mobility (A) KDM3A knock-down with shRNA in HeLa cells, and scratch-mobility analysis with or without IL-6 treatment. Scratch closure was monitored in HeLa cells for 48 hours at 24 hour intervals. Cell migration (%) was quantified by calculating the scratch width as shown in the right panel graph. *** P <0.001 (Student's t -test). (B) KDM3A was knocked down with shRNA in HeLa cells, and cell proliferation according to the presence or absence of IL-6 treatment was monitored and confirmed every 6 hours. The cell culture area was calculated as shown in the graph to quantify the proliferation efficiency (%). *** P <0.001 (Student's t -test). (C) Representative image of cells stained with BrdU. The percentage of increased BrdU positive (BrdU +) cells after IL-6 treatment decreased after knock-down of KDM3A by shRNA. After knocking down KDM3A with shRNA in HeLa cells, transfecting resistant KDM3A WT ^R or YA ^R and confirming BrdU positive (BrdU +) cells with or without IL-6 treatment by immunostaining. Nuclei stained with DAPI. Scale bar, 10 μm. *** P <0.001 (Student's t -test). (D) Analysis of colony formation of cells according to whether KDM3A is knocked down with shRNA in HeLa cells, and MYC is transfected together, and whether IL-6 is processed. Cells were fixed and stained with crystal violet solution. The number of colonies was quantified as shown in the graph. *** P <0.001 (Student's t -test). (E) Schematic model depicting the JAK2-KDM3A-STAT3 signal transduction axis. Regulation of H3K9 demethylation induced by JAK2-induced KDM3A phosphorylation is one of the most important epigenetic phenomena in the transcriptional regulation of STAT3 target genes.

This application is based on the discovery that KDM3A acts as a major epigenetic factor for JAK2-STAT3 activation in cancer cells. Specifically, KDM3A is phosphorylated at residue 1101 by JAK2, and phosphorylated KDM3A acts as a co-activator for transcriptional activation of a STAT3 target gene, such as MYC , with a decrease in demethylation activity for histone 3 protein (H3K9me2). It was found to increase the proliferation of.

This indicates that inhibition of KDM3A phosphorylation may serve as a target for the development of new therapeutic agents that inhibit cancer development and / or progression that control the carcinogenic effects of the JAK2-STAT3 signaling pathway.

Thus, in one aspect, the present application relates to a method of screening for a cancer therapeutic or cell proliferation inhibitor acting on the JAK2-STAT3 pathway, which is identified herein, which plays an important role in the development and / or progression of cancer.

In one embodiment, the method is a method for screening a substance for treating cancer related to a signal transducer and activator of transcription 3 (JAS2) -STAT (STAT) signaling pathway, and the method includes the following steps: JAK2 protein And a KDM3A (lysine-specific demethylase 3A) protein comprising the 1101 th tyrosine residue based on the sequence of SEQ ID NO: 1 that can be phosphorylated by the JAK2 protein is expected to inhibit phosphorylation of the tyrosine residue by the JAK2. A first step of culturing in the presence of a test substance; A second step of measuring the phosphorylation degree of the tyrosine residue of the KDM3A protein; And a third step of selecting as a candidate substance capable of inhibiting the growth of cancer cells when phosphorylation of the KDM3A protein decreases when treated with a test substance, compared to a control group not treated with a test substance as a result of the measurement.

Although the relevance of the JAK2-STAT3 pathway to cancer and the relevance of KDM3A to cancer are known, the function of KDM3A as an essential epigenetic gene for activation of the JAK2-STAT3 signaling pathway was first identified herein. The specifically activated JAK2-KDM3A signaling cascade provides a functional and mechanical link between activation of the JAK2-STAT3 signaling pathway and epigenetic control by bringing about a major epigenetic event, H3K9 methylation.

JAK2 (Janus tyrosine kinase 2) signal transducer and activator of transcription 3 (STAT3) signaling pathways are essential pathways for cell development, cell differentiation, and homeostasis regulation. Specifically, JAK2 is a non-receptor tyrosine phosphorylase that induces the cytoplasmic signal transduction process, and JAK2 is also present in the nucleus of human hematopoietic cells and is known to act as a direct phosphorylase of histone H3Y41 (tyrosine residue 41 of histone 3). When JAK2 is activated, it activates transcription factor STAT3. STAT3 is phosphorylated by JAK2 and forms a stable homodimer or heterodimer to induce local transcriptional activation of the target gene. This abnormal response of the JAK2-STAT3 signaling pathway has been shown to induce abnormal cell proliferation, migration and anti-cell death in various types of cancer, including cervical cancer, ovarian cancer, gastric cancer and breast cancer. (Griffiths DS , et al. (2011) LIF-independent JAK signaling to chromatin in embryonic stem cells uncovered from an adult stem cell disease.Nature cell biology 13 (1): 13-21 .; Marotta LL , et al. (2011 ) The JAK2 / STAT3 signaling pathway is required for growth of CD44 (+) CD24 (-) stem cell-like breast cancer cells in human tumors.The Journal of clinical investigation 121 (7): 2723-2735 .; Yu H, Kortylewski M, & Pardoll D (2007) Crosstalk between cancer and immune cells: role of STAT3 in the tumour microenvironment.Nature reviews.Immunology 7 (1): 41-51.).

We have identified that KDM3A serves as an epigenetic factor essential for activation of the JAK2-STAT3 signaling pathway. Specifically, KDM3A is phosphorylated at the 1101 th tyrosine residue based on the sequence of SEQ ID NO: 1 by JAK2, and phosphorylated KDM3A is a STAT3 target gene such as MYC with a decrease in H3K9me2 (histone 3 where lysine at the 9th residue is methylated). It was found that it acts as a STAT3-dependent co-activator for the transcriptional activation of cancer cells and increases the proliferation of cancer cells.

KDM3A (Lysine (K) -Specific Demethylase 3A) is a protein that demethylates H3K9me1 or H3K9me2 (one or two methylation at the ninth lysine residue of histone 3) and is overexpressed in many cancers including bladder cancer and lung cancer. (Hyun-Soo Cho et al. International Journal of Cancer Int. J. Cancer: 131, E179-E189 (2012) VC 2011 UICC).

The JAK2, STAT3 and KDM3A gene and protein sequences used in the method according to the present application are known, for example the human protein sequence is KDM3A: NP_060903.2; JAK2: NP_001309123.1; STAT3: NP_644805.1; HISTONE 3: AAN39284.1, known to the NCBI. It will be apparent to those skilled in the art that once the protein sequence is known, the nucleic acid sequence can be easily derived therefrom.

In one embodiment, each protein sequence is disclosed herein as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

In the method according to the present application, as long as it has the above-described functions, full-length or fragments having various origins and protein sequences of each origin and sequences substantially identical thereto may be used. Substantially the same applies to all Iranian, protein and nucleic acid sequence levels, and may have one or more substitutions, deletions, or additions to base or amino acid residues compared to the reference or reference sequence, but the overall difference in function It means having a level of functionality that does not or does not degrade the function. Homology aligns the target sequence to the maximum possible correspondence, and when the aligned sequence is analyzed using an algorithm commonly used in the art, at least 61% homology, more preferably 70% phase Refers to a sequence that exhibits homology, even more preferably 80% homology, most preferably 90% or more, particularly 95% or more. Alignment methods for sequence comparison are known in the art. See, for example, Smith and Waterman, Adv. Appl. Math. (1981) 2: 482 ; Needleman and Wunsch, J. Mol. Bio. (1970) 48: 443; Pearson and Lipman, Methods in Mol. Biol. (1988) 24: 307-31; Higgins and Sharp, Gene (1988) 73: 237-44; Higgins and Sharp, CABIOS (1989) 5: 151-3; Corpet et al., Nuc. Acids Res. (1988) 16: 10881-90; Huang et al., Comp. Appl. BioSci. (1992) 8: 155-65 and Pearson et al., Meth. Mol. Biol. (1994) 24: 307-31. NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. (1990) 215: 403-10) is accessible from NBCI, etc., such as blast, blastp, blasm, blastx, tblastn and tblastx It can be used in conjunction with a sequence analysis program. BLSAT can be accessed at www.ncbi.nlm.nih.gov/BLAST/, and a method for comparing sequence homology using this program can be found at www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

In one embodiment, the protein may be provided in the form of cells expressing it.

For example, a mammalian cell expressing a protein (transient or stable transfer or endogenous expression), for example, but not limited to, the protein expressing and acting cancer cells may be used, including humans Either from an animal such as a mammal or an established cell line can be used.

In one embodiment, eukaryotic cells are used. In other embodiments, cancer cells or cancer cell lines are used, for example, Hela or HEK293 cell lines may be used. The cell line may be used by transferring the plasmid expressing the protein using a known method or a method described in Examples herein.

In the method according to the present application, when the JAK2-STAT pathway is activated in the presence of a stimulus, JAK2 phosphorylates residue 1101 of the KDM3A protein. Thus, in one embodiment, in the method according to the present application, inhibition of the JAK2-STAT3 signaling pathway can be measured by reducing or inhibiting phosphorylation of the KDM3A protein by the JAK2.

In the method according to the present application, the degree of phosphorylation inhibition can be measured using a known method, for example, can be confirmed using a protein blot method. In one embodiment, antibodies can be used. Antibodies that can specifically recognize the phosphorylated 1101 residue of the KDM3A protein can be used. In addition, a known antibody that does not specifically recognize the phosphorylated 1101 residue can be used as a control or comparative bacterium. Compared to the control group (when the test substance is not treated), one that suppresses the phosphorylation degree of the KDM3A protein can be selected as a candidate for treatment. In this case, the protein can be labeled using a variety of markers for ease of detection, such as protein tags, biotin, fluorescent substances, acetylation, methods known in the art, such as radioisotopes, or commercially available protein labeling kits. It can be detected using a detector suitable for the labeled substance.

In addition, since the KDM3A demethylates H3K9, inhibition of the JAK2-STAT signaling pathway can also be measured by reducing the demethylation activity of KDM3A for methylated H3K9, i.e., reducing the demethylation of methylated H3K9.

Detection of methylation or demethylation in the method according to the present application can be performed using known methods, including, but not limited to, the use of antibodies specifically recognizing methylated proteins or demethylated proteins. , See also examples herein.

In addition, since KDM3A phosphorylated by JAK2 has activity as a cofactor for STAT3 protein, inhibition of the JAK2-STAT signaling signaling pathway can also be measured by a decrease in the transcriptional activity of the STAT3 protein. STAT3 protein is a transcription factor (Transcription Factor) that binds to a promoter capable of binding to STAT and promotes expression of a gene operably linked thereto into transcription RNA, and phosphorylated KDM3A acts as a cofactor in this process.

The activity as a cofactor for the STAT3 protein in the method according to the present application can thus be measured by the transcriptional activity of the STAT3 protein, and this method can be performed in consideration of the examples and technical knowledge of the present application. For example, a plasmid operably linked to the STAT3 promoter to a target gene, such as Myc, can be used, and transcriptional activity from the plasmid can be detected through various methods for determining the amount of target mRNA, for example, reverse transcription PCR. It can, but is not limited to.

In the method according to the present application, the JAK2-STAT signaling pathway can be activated. JAK2-STAT signaling pathway can be activated, especially when the method according to the invention is performed in cells. In one embodiment, a substance acting on activation of the JAK2-STAT signaling pathway mediated by the glycoprotein 130 (gp130) receptor is used, and a substance sharing the gp130 mediated signaling molecule is similar to activating the JAK2-STAT signaling pathway. Function. Sriram K et al. , The Journal of Biological Chemistry 279, 19936-19947, 2004). Therefore, various known substances having the above effects can be used for activation, and IL (Interleukin) -6, IL-11 or Oncostatin M (Osm) can be used. have. In other embodiments, IL-6 can be used.

In the method according to the present application, treatment with a test substance expected to inhibit the JAK2-STAT pathway via KDM3A. Inhibition of this pathway can be measured by one or more methods of reducing or inhibiting phosphorylation of KDM3A protein, reducing or inhibiting transcriptional activity of STAT3 protein, reducing or inhibiting demethylation of H3K9 protein, as described above. Means inhibition or reduction as compared to a control not treated with a test substance, and those skilled in the art will readily be able to determine the degree of inhibition or reduction based on the content disclosed herein and / or common knowledge in the art.

The test materials used in the methods herein will act on the JAK2-STAT pathway signaling system to reduce phosphorylation of KDM3A, thereby inhibiting the activity of a substance or STAT3 protein that is expected to specifically inhibit the demethylation of H3K9. Expected low molecular weight compounds, high molecular weight compounds, mixtures of compounds (e.g. natural extracts or cell or tissue cultures), or biopharmaceuticals (e.g. proteins, antibodies, peptides, DNA, RNA, antisense oligonucleotides, RNAi, aptamers) , RNAzyme and DNAzyme) or sugars and lipids.

In one embodiment according to the present application, a low molecular compound may be used as a test substance. The test substance can be obtained from a library of synthetic or natural compounds and methods for obtaining a library of such compounds are known in the art. Synthetic compound libraries are available from Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA) and Sigma-Aldrich (USA), and libraries of natural compounds are available from Pan Laboratories (USA) and Available from MycoSearch (USA). Test materials can be obtained by a variety of combinatorial library methods known in the art, e.g., biological libraries, spatially addressable parallel solid phase or solution phase libraries, deconvolution It can be obtained by the required synthetic library method, the "1-bead 1-compound" library method, and the synthetic library method using affinity chromatography screening. Methods for the synthesis of molecular libraries are described in DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al., J. Med. Chem. 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al., J. Med. Chem. 37, 1233, 1994 and the like. For example, for the purpose of screening a drug, a compound having a low molecular weight therapeutic effect may be used. For example, a compound having a weight of about 1000 Da, such as 400 Da, 600 Da or 800 Da, may be used. Depending on the purpose, these compounds can form part of a library of compounds, and the number of compounds that make up the library can vary from dozens to millions. Such compound libraries include peptides, peptoids and other cyclic or linear oligomeric compounds, and low molecular compounds based on templates, such as benzodiazepine, hydantoin, viaryl, carbocycle and polycycle compounds (e.g. naphthalene, phenothi) Azine, acridine, steroid, etc.), carbohydrate and amino acid derivatives, dihydropyridine, benzhydryl and heterocycles (such as triazine, indole, thiazolidine, etc.), but this is merely exemplary It is not limited to this.

Biologics can also be used for screening, for example. Biologics refers to a cell or a biomolecule, and a biomolecule refers to a substance produced by using a protein, nucleic acid, carbohydrate, lipid, or cell system in vivo and ex vivo. Biomolecules may be provided alone or in combination with other biomolecules or cells. Biomolecules include, for example, polynucleotides, peptides, antibodies, or other proteins or biological organic substances found in plasma.

As a result of the experiment, a substance that inhibits the reduction of phosphorylation of KDM3A, demethylation of H3K9, or a substance that inhibits activity as a cofactor of STAT3 protein in the presence of the test substance is selected as a candidate substance as compared to a control group not in contact with the test substance. About 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100% or more or a decrease in this abnormality may be selected as a candidate material.

Proteins as used herein can be prepared using methods known in the art. In particular, it uses genetic recombination technology. For example, a plasmid containing a corresponding gene encoding the protein may be transferred to a prokaryotic or eukaryotic cell, for example, an insect cell or a mammalian cell, and then overexpressed and purified. The plasmid can be used, for example, by transfecting an animal cell line as used in the exemplary embodiment of the present application, and then purifying the expressed protein, but is not limited thereto. In this case, the protein can be labeled using a variety of markers for convenience of detection, such as biotin, fluorescent substance, acetylation, radioisotope, or a commercially available protein labeling kit. It can be detected using a detector suitable for.

Alternatively, the DNA or RNA sequence encoding the screening protein is expressed in an appropriate host cell to make the cell lysate, or the mRNA of the screening protein is translated in vitro, and then the screening protein can be purified by a protein separation method known in the art. Can be. Usually, to remove cell debris, etc., the cell lysate or the in vitro translated product is centrifuged, and then precipitated, dialysis, and various column chromatography are applied. Ion exchange chromatography, gel-permeation chromatography, HPLC, reverse phase-HPLC, preparation SDS-PAGE, affinity columns, etc. are examples of column chromatography. Affinity columns can be made, for example, using anti-screening protein antibodies.

Other types of test substances used in the above method may be referred to the aforementioned.

Candidates selected by the method according to the present application can be developed / used as an inhibitor of unregulated cell growth or as a therapeutic agent for cancer.

Cancers caused by JAK-STAT signaling abnormalities are not limited herein, but are solid cancers, particularly cervical cancers.

As used herein, the term “treatment” is a concept that includes inhibition, elimination, alleviation, alleviation, amelioration, and / or prevention of a disease or symptom or condition resulting from a disease.

In another aspect, the present application relates to an antibody that specifically recognizes KDM3A phosphorylated with the 1101 th tyrosine residue.

In this application, as shown in D of FIG. 1 of the present application, KDM3A is specifically phosphorylated at lysine residue 1101, and the antibody according to the present application has been shown to specifically recognize the phosphorylated KDM3A without cross-reactivity. KDM3A, whose residue has been transformed to tyrosine, is not recognized, and thus has excellent specificity.

The antibody of the present invention includes a complete antibody, an antigen-binding fragment of an antibody molecule, and an antibody or fragment functionally equivalent thereto. In addition, the antibody of the present invention may be of IgG, IgM, IgD, IgE, IgA or IgY type, and may be of IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2 class or subclass thereof. Antibodies of the invention include monoclonal, polyclonal, chimeric, short chain, bispecific, anthropogenic and humanized antibodies and active fragments thereof.

A complete antibody is a structure having two light chains and two heavy chains, and each light chain is connected by a heavy chain and a disulfide bond. The heavy chain constant regions have gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and subclasses gamma 1 (γ1), gamma 2 (γ2), and gamma 3 (γ3) ), Gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant region of the light chain has kappa (κ) and lambda (λ) types (Cellular and Molecular Immunology, Wonsiewicz, MJ, Ed., Chapter 45, pp. 41-50, WB Saunders Co. Philadelphia, PA (1991); Nisonoff, A., Introduction to Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, Sinauer Associates, Inc., Sunderland, MA (1984)).

Examples of antigen-binding fragments or active fragments of antibodies that bind antigens include Fv, ScFv, Fab, Fab 'and F (ab') 2. Among antibody fragments, Fab has a structure having a variable region of a light chain and a heavy chain, a constant region of a light chain, and a first constant region (CH1) of a heavy chain, and has one antigen-binding site. Fab 'differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. The F (ab ') 2 antibody is produced by cysteine residues in the hinge region of Fab' forming disulfide bonds. sFv is a minimal antibody fragment having only a heavy chain variable region and a light chain variable region, and a recombinant technique for generating an Fv fragment is disclosed in PCT International Patent Application Publication Nos. WO 88/10649, WO 88/106630 and the like. In the double-chain Fv (two-chain Fv), the heavy chain variable region and the light chain variable region are connected by a non-covalent bond, and in sFv (single-chain Fv), the variable region of the heavy chain and the variable region of the single chain are commonly covalently linked through a peptide linker. Or directly connected at the C-terminus to form a dimer structure such as a double chain Fv. These antibody fragments can be obtained using proteolytic enzymes (e.g., restriction digestion of the whole antibody with papain yields Fab and cleavage with pepsin yields F (ab ') 2 fragment), gene It can be produced through recombinant technology. For details on this method, see, eg, Khaw, BA et al. J. Nucl. Med. 23: 1011-1019 (1982); Rousseaux et al. Methods Enzymology, 121: 663-69, Academic Press (1986).

The term “functionally equivalent antibody” is an antibody capable of specifically recognizing phosphorylated KDM3A comprising at least one major functional property, ie, other mutations in which the 1101th residue exhibits phosphorylated KDM3A effect, of any of the aforementioned types. , Including classes or fragments, including antibodies produced by various methods such as phage display, any individual and cell, such as bacteria, insect cells, mammalian cells, etc., to produce the desired antibody.

Antibodies of the invention can be obtained by animal serum, cell culture or recombinant DNA technology.

In one embodiment of the present invention, the antibody of the present invention is a polyclonal antibody, and methods for preparing the polyclonal antibody are known in the art.

In one embodiment of the invention the antibody of the invention is a monoclonal antibody. Methods for the production of monoclonal antibodies are known in the art and can be prepared through traditional cloning and cell fusion methods. Typically, the antigen is administered to a transgenic mouse engineered to produce a wild-type mouse or in-breed mouse (BALB / c or C57BL / 6), rat, rabbit or other mammalian or wild-type or human antibody (eg subcutaneous or intraperitoneal). Administration). The antigen can be administered alone or in combination with an adjuvant, and the antigen can be expressed in a vector or introduced in the form of a fusion protein capable of inducing DNA or an immune response. Fusion proteins include peptides capable of inducing an immune response, including carrier proteins such as beta-galactosidase, glutathione, S-transferase, keyholelimpet hemocyanin (KLH) and fetal bovine serum. . After boosting the animal 2-3 times with these antibodies, the spleen is expelled and fused with a myeloma cell line (e.g., SP2 / O from ATCC, USA) in the presence of a fusion accelerator such as polyethylene glycol to generate a hybridoma cell line. And for more details, see Kohler and Milstein. Nature 256: 495-497 (1975) and Harlow and Lane. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988).

Hereinafter, examples are provided to help understanding of the present invention. However, the following examples are provided only for easier understanding of the present invention, and the present invention is not limited to the following examples.

Example

Experiment method and materials

Cell culture and reagents

Cells were cultured in an environment of 37 ° C., 5% (v / v) CO ₂ . HEK293T and HeLa cells were incubated with 10% FBS (Gibco) in Dulbecco's modified Eagle's medium (DMEM, Welgene) with penicillin (100 U / mL) and streptomycin (100 μg / mL). IL-6 (R & D system, 50 NG / mL), OSM (R & D system, 20 NG / mL), IL-11 (R & D system, 5 NG / mL) AG490 (Sigma 10 μM) TG101348 (Selleckchem, 2.5 μM), Asciminib (Selleckchem, 250 nM) or PP2 (Sigma, 10 μM) was treated for the indicated time. JAK1 (# 3332), JAK2 (# 3230), phospho-STAT3 (# 9131) and H3K9me2 (# 9753, # 4658, ab1220) (Cell Signaling Technology, Abcam); STAT3 (sc-8019) (Santa Cruz); KDM3A (nb100-77282) (Novus); phospho-Tyrosine 4G10 (05-1050) (Millipore); HA (MMS-101R) (Covance); And FLAG (F3165, Sigma).

Antibodies that specifically recognize KDM3A phosphorylated with the 1101 th residue herein were first prepared herein. The antibody was prepared in rabbits using 4 mg of peptide KM-pY-NAYGLI (a peptide in which the 1101 th tyrosine residue was phosphorylated based on SEQ ID NO: 1) as an antigen, and was prepared by requesting Peptron (Korea).

Reporter analysis

Using the Lucifer Raise System (Promega), M67 (4X-STAT response factor) -lucifer raise activity was measured using a luminometer 48 hours after transfection and normalized to β-galactosidase activity. At least three independent experiments were performed.

Quantitative RT-PCR

For qRT-PCR analysis, total RNA was extracted using Trizol (Invitrogen) and reverse transcribed using RevertAid ™ Reverse Transcriptase (Enzynomics). The following primers were used for the PCR reaction: hMYC: forward 5'-GAAGACGGGCTCCTTTGA-3 ', reverse 5'-CAGCACTAAGCTCTGATACA-3'; hHPRT; Forward 5'-TGACACTGGCAAAACAATGCA-3 ', reverse 5'-GGTCCTTTTCACCAGCAAGCT-3'; MRNA was detected and analyzed by an ABI Prism 7300 system with SYBR Green (Molecular Probes).

ChIP assay

The following ChIP primers for the STAT binding elements of hMYC were used in PCR reactions: forward 5'-GCCTCTGGCCCAGCCCTCCCGCTGAT-3 ', reverse 5'-GCAAAGTGCCCGCCCGCTGCTATGG-3'.

Identification of phosphorylation sites by LC-MS / MS

Tryptic digest of KDM3A was analyzed by nanoelectrospray LC-MS / MS. High pressure liquid chromatography separation was performed on an Ultimate instrument equipped with Famous Autosampler (LC Packings). The column was composed of fused silica capillaries having an outer diameter of 360 μm and an inner diameter of 75 μm, and a length of 15 cm was used using a 300-mm C18 beads (Grace Vydac) having a diameter of 5 μm. The exit of the nanocolumn was connected inline to a distal coated silica PicoTip needle (New Objective). The solvent system consisted of solvent A (0.1% formic acid and 5% acetonitrile) and solvent B (0.1% formic acid and 90% acetonitrile). The gradient was linear in 0-40% solvent B over 50 minutes, then 90% solvent B for 5 minutes. Tandem mass spectra were recorded on an API QSTAR Pulsar Q-TOF mass spectrometer (Applied Biosystems) in an information dependent acquisition mode. The MS / MS spectrum was used to search the National Center for Biotechnology Information and the expressed sequence tag database using Mascot (Matrix Science). The identified phosphorylated peptides were manually verified.

Immune cell staining

HeLa cells were cultured on a cover slip and washed with PBS. Cells were fixed with 2% paraform aldehyde for 10 minutes at room temperature and made permeable three times with 0.5% Triton X-100. After blocking, Flag, JAK2 and H3K9me2 antibodies were labeled with Alexa Fluor 594 and 488 mouse and rabbit IgG labeling kits (Molecular Probe), respectively.

In vitro cell motility assay

Wound healing scratching motility assay was performed on pLKO.1 control vector or HeLa cells transfected with pLKO.1-shKDM3A. Cells were seeded in 6-well culture plates and cultured until confluence. Cells were scraped with a 200 μl micropipette tip and incubated at 37 ° C. for 48 hours. Micrographs of closed gaps were taken at 0, 24 and 48 hours using an EVOS xl transmission light microscope (AMG, Bothell, WA). Cell travel distance was quantified by gap distance using Graph Prism software.

Cell proliferation assay

The pLKO.1 vector or pLKO.1-shKDM3A was transfected into HeLa cells with lipofectamine reagent. One day after transfection, the presence or absence of IL-6 treatment was separated, and the cells were separated and cultured in a 24-well plate. Cell proliferation was counted for each 6 hour using JuLiTM stage live imaging equipment (NanoEnTek Inc) and analyzed with JuLiTM STAT software according to the manufacturer's guide.

Statistical analysis

Statistical differences between test and control samples were determined by Student's t- test. ANOVA performed comparisons of several samples. The bar graph of the graph represents the mean ± sem. Graphpad Prism software was used for all analysis.

Example 1. Phosphorylation of tyrosine 1101 residue of KDM3A by JAK2

According to liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS) analysis, KDM3A was phosphorylated to the tyrosine 1101 (Y1101) residue (FIG. 1A). Interestingly, the Y1101 residue and the surrounding amino acids of KDM3A are well conserved between species (Figure 1B). Coimmunoprecipitation (Co-IP) analysis using phosphorylated tyrosine specific antibodies shows that KDM3A is phosphorylated to tyrosine residues (FIG. 1C). Therefore, we prepared an antibody specific for KDM3A phosphorylated at residues Y1101 (KDM3A-Y1101p) and confirmed that cross-reaction does not occur and efficacy by immunoblot analysis (FIG. 1D). Using the KDM3A-Y1101 phosphorylation antibody, we found that in the case of the Y1101A (YA) mutant form of KDM3A where the 1101th amino acid residue tyrosine was replaced by alanine, tyrosine phosphorylation of KDM3A did not appear (Figure 1E). These data indicate that KDM3A is phosphorylated at the Y1101 tyrosine residue.

Next we decided to explore the top tyrosine phosphorylases responsible for KDM3A phosphorylation. Because KDM3A is mainly localized in the nucleus, we sought tyrosine phosphorylases in the nucleus. We hypothesized that JAK1, JAK2, Src, and Abl1 are present in the nucleus and cytoplasm, so they can phosphorylate KDM3A in the nucleus. Immunoprecipitation data confirmed that KDM3A binds to both JAK1 and JAK2 in the presence of IL-6, a well-known high-level physiological signal for JAK activation and nuclear transfer (Figures 1F and G). Next, we investigated whether JAK1 or JAK2 activated by IL-6 treatment induces KDM3A phosphorylation. We found that JAK2 increased KDM3A tyrosine phosphorylation after IL-6 treatment, but confirmed that KDM3A phosphorylation was not increased by JAK1 (FIG. 1H). This indicates that phosphorylation of the KDM3A Y1101 tyrosine residue is a JAK2 specific action. In addition, although the phosphorylation of KDM3A was induced when confirmed with the KDM3A-Y1101 phosphorylation antibody after IL-6 treatment, it was confirmed that KDM3A phosphorylation induced by JAK2 was broken by IL-6 treatment when JAK2 was knocked down (FIG. 1I ).

To determine whether JAK2 enzyme activity is important for KDM3A phosphorylation, wild type (WT) of JAK2, continuously activated V617F mutant (CA), and phosphorylation-inactive mutant (DN) were analyzed in HeLa cells. The JAK2 WT and CA mutants showed phosphorylation activity against KDM3A in both in vivo (Figure 1J) and in vitro (Figure 1K), whereas the DN mutant did not. Indeed, it was confirmed that KDM3A phosphorylation induced by IL-6 disappeared when treated with JAK2 inhibitors AG490 and TG101348 (FIGS. 1L and M), but Src strain inhibitor PP2 (FIG. 1N) or Abl inhibitor Asciminib (FIG. 10). Phosphorylation hardly disappeared with other inhibitors. Taken together, these data confirmed that KDM3A is phosphorylated to tyrosine by JAK2 in the nucleus, both in vivo and in vitro.

Example 2. Increase of demethylase activity by KDM3A phosphorylation induced by IL-6

Although JAK2 is known to exclude HP-1α from the STAT target gene for transcriptional activation in human hematopoietic cells, it has not been determined whether JAK2 signaling modulates KDM3A-dependent H3K9 demethylation (transcriptional activity marker). Therefore, in the present invention, it was confirmed that JAK2-dependent phosphorylation of KDM3A is induced by IL-6 treatment to confirm whether the degree of methylation of H3K9me2 changes during IL-6 treatment. Indeed, H3K9me2 was reduced during IL-6 treatment in HeLa and HEK293T cells with increased KDM3A phosphorylation and STAT3 phosphorylation (Figure 2A). Next, it was investigated whether JAK2 induced by IL-6 was responsible for H3K9me2 level reduction. In fact, when IL-6 treatment, the level of H3K9me2 decreased, but when JAK2 was knocked down by shRNA, the reduction of H3K9me2 level did not appear even after IL-6 treatment, immunoprecipitation (Fig. 2B) and immunostaining assay (FIG. 2C).

We also investigated whether the demethylation of H3K9 mediated by JAK2 depends on KDM3A. A decrease in JAK2-dependent H3K9me2 level by IL-6 treatment was not observed when KDM3A was knocked down (FIG. 2D). To test whether phosphorylation of KDM3A by JAK2 affects intrinsic histone demethylation activity, KDM3A knock-down cells were transfected with a shRNA resistant form of KDM3A WT ^R or YA ^R mutation. Interestingly, it was confirmed by immunostaining assay that the YA mutant of KDM3A significantly reduced the enzyme activity on H3K9me2 compared to WT of KDM3A (FIG. 2E). In addition, when KDM3A WT ^R or YA ^R mutation was introduced into KDM3A knock-down cells depending on whether IL-6 was treated, the level of H3K9me2 was decreased in KDM3A WT only in the case of IL-6 treatment, but not in the YA mutant. (Figure 2F).

In addition, it was investigated whether cytokines other than IL-6 were treated to induce KDM3A phosphorylation with a decrease in H3K9me2 levels. Indeed, KDM3A phosphorylation was also induced with demethylation of H3K9me2 by Oncostatin M (OSM) and IL-11, other stimulation signals of STAT3 activation (Figures 2G and H). Since IL-6, IL-11 and OSM share gp130 mediated signaling molecules as receptors, they have been shown to have similar effects on KDM3A phosphorylation. However, it is impossible to rule out the possibility that a specific mode of action and a sub-target gene can be differentially regulated by each STAT3 activation stimulus. These data indicate that JAK2-dependent tyrosine phosphorylation of KDM3A induces enhanced enzymatic activity, thereby reducing H3K9me2 levels.

Example 3 Characterization of KDM3A as a transcriptional co-activator in the JAK2-STAT3 signaling pathway

JAK2 activates the STAT3 target gene by interacting with STAT3. Activated JAK2 migrates to the nucleus to induce STAT3 phosphorylation for transcriptional activation. In addition, JAK2 entering the nucleus is known to function as a tumor-inducing factor by acting on other specific substrates such as histones other than STAT3 in the nucleus. To test whether phosphorylated KDM3A can function as a transcriptional coactivator of the JAK2-STAT3 signaling pathway, it was first investigated whether KDM3A binds to STAT3. It was confirmed through immunoprecipitation that binding between KDM3A and STAT3 was significantly improved by IL-6 treatment (FIG. 3A).

To investigate whether KDM3A binds to the JAK2-STAT3 target promoter through STAT3, chromatin immunoprecipitation (ChIP) analysis was performed on the MYC promoter containing a functional STAT binding site. Through the ChIP analysis, it was confirmed that JAK2, phosphorylated STAT3 and phosphorylated KDM3A were all present in the MYC promoter with a decrease in H3K9me2 level according to IL-6 treatment (FIG. 3B). These results indicate that phosphorylated KDM3A is directly involved in the transcriptional activation of the JAK2-STAT3 target gene induced by IL-6. In addition, it was confirmed that the level of MYC mRNA and protein induced by IL-6 was significantly reduced through knock-down of KDM3A by shRNA (FIGS. 3C and D). In addition, when KDM3A WT and YA mutants of shRNA-resistant form were introduced after knock-down of KDM3A, it was confirmed that the target MYC mRNA and protein level of STAT3 increased when IL-6 treatment was performed only when KDM3A WT was introduced. (Figures 3E and F).

In addition, in the analysis using the M67 promoter-luciferase reporter containing a functional STAT3 response element, KDM3A phosphorylation increases the M67 promoter-luciferase reporter activity in the presence of the persistent active form of STAT3 (STAT3 CA), but the activity of STAT3 is not seen. In the non-form (STAT3 DN), it failed to bind to the STAT3 response element (FIG. 3G). STAT3-dependent luciferase reporter activity was attenuated by KDM3A knock-down (Figure 3H), but increased by KDM3A overexpression (Figure 3I). Taken together, these data indicate that KDM3A function is important for the expression of the target gene of JAK2-STAT3, which is activated by IL-6 as a transcriptional co-activator of STAT3.

Example 4 Increased cell proliferation and mobility by KDM3A phosphorylation and offset effect by KDM3A knockdown

We investigated whether KDM3A phosphorylation by JAK2 activated by IL-6 is involved in regulating cancer cell growth, proliferation, mobility, and colony formation, because dysregulation of the JAK2-STAT3 signaling system is associated with malignant tumor induction in humans. Cell mobility analysis according to the presence or absence of treatment with IL-6 that induces KDM3A phosphorylation was performed. It was confirmed that the mobility was increased by IL-6 treatment, and the interval was narrowed after about 48 hours. On the other hand, it was confirmed that the cell mobility of KDM3A shRNA is significantly reduced in cells in which KDM3A is knocked down (FIG. 4A). These data indicate that KDM3A is involved in the enhancement of cell mobility in the treatment of IL-6.

Next, cell growth efficiency was measured through cell growth curve analysis and BrdU staining and colony formation analysis. In the cell proliferation efficiency, similar results were obtained in the case of cell mobility analysis. Cell growth and BrdU binding were increased when IL-6 treatment was performed, but when KDM3A was knocked down, cell growth and BrdU binding were significantly decreased even when IL-6 was treated (FIGS. 4B and C). When KDM3A WT ^R and YA ^R mutations were introduced in KDM3A knockdown cells, it was confirmed that the increase in BrdU binding was restored upon IL-6 treatment only in the case of KDM3A WT ^R (FIG. 4C). The effect of MYC introduction on colony formation with and without IL-6 treatment and with or without KDM3A knockdown was analyzed. When MYC was introduced from KDM3A knock-down cells, colony-forming ability was decreased upon IL-6 treatment (FIG. 4D).

These data potentially suggest through increased colony formation that the expression of MYC activated by KDM3A upon IL-6 treatment can induce cancer progression. Collectively, these data indicate that phosphorylated KDM3A promotes cell growth mediated by IL-6, thereby regulating cell survival and cancer progression (FIG. 4E).

In the present invention, it was found that KDM3A provides a functional and mechanical link between intracellular JAK2-STAT3 signaling to the upper stimulus signal and lower epigenetic genetic mechanism. It was also suggested that the regulation of H3K9 demethylation by tyrosine phosphorylated KDM3A induced by JAK2 is one of the main epigenetic phenomena that induces proliferation and mobility of cancer cells. The results herein identified the JAK2-KDM3A-H3K9me2 signal transduction axis in the nucleus induced by IL-6 that has never been reported previously, and that this signaling pathway increases cancer cell mobility, proliferation and growth. JAK2 activation allows phosphorylated KDM3A to bind strongly to STAT3 and act as a co-activator of STAT3 through increased H3K9 demethylase function. It is known that dysregulation of the JAK2-STAT3 signaling system has serious consequences, such as degenerative diseases and cancer, because the JAK2-STAT3 signaling pathway is evolutionarily well preserved in maintaining cell homeostasis. Therefore, signal-dependent regulation of epigenetic modulators such as KDM3A and identification of their dysregulation can provide new insights into the mechanism of cancer progression and open a challenging path for new drug development.

More than 30 KDM families with a Jumanji (JmjC) domain are known. For the past 10 years, the development of clinical target candidates has not been significant for KDM containing the Jumanji (JmjC) domain, but four compounds are already in clinical trials for other demethylating enzymes, such as LSD1. The major obstacles to the development of selective compounds that modulate the Jumanji (JmjC) domain-containing KDM are high structural similarity and potential functional redundancy between family members. Since we have presented a new signaling pathway for JAK2-KDM3A-H3K9me2, we may be able to provide a new treatment strategy through regulation of an important carcinogenesis signaling pathway targeting KDM3A phosphorylation by JAK2.

Although the exemplary embodiments of the present application have been described in detail above, the scope of the present application is not limited thereto, and various modifications and improvements of those skilled in the art using the basic concept of the present application defined in the following claims are also provided. It belongs to.

All technical terms used in the present invention, unless defined otherwise, are used in a sense as commonly understood by those skilled in the art in the relevant field of the present invention. The contents of all publications described by reference herein are incorporated into the present invention.

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(1312) <223> KDM3A <400> 1 Met Val Leu Thr Leu Gly Glu Ser Trp Pro Val Leu Val Gly Arg Arg   1 5 10 15 Phe Leu Ser Leu Ser Ala Ala Asp Gly Ser Asp Gly Ser His Asp Ser              20 25 30 Trp Asp Val Glu Arg Val Ala Glu Trp Pro Trp Leu Ser Gly Thr Ile          35 40 45 Arg Ala Val Ser His Thr Asp Val Thr Lys Lys Asp Leu Lys Val Cys      50 55 60 Val Glu Phe Asp Gly Glu Ser Trp Arg Lys Arg Arg Trp Ile Glu Val  65 70 75 80 Tyr Ser Leu Leu Arg Arg Ala Phe Leu Val Glu His Asn Leu Val Leu                  85 90 95 Ala Glu Arg Lys Ser Pro Glu Ile Ser Glu Arg Ile Val Gln Trp Pro             100 105 110 Ala Ile Thr Tyr Lys Pro Leu Leu Asp Lys Ala Gly Leu Gly Ser Ile         115 120 125 Thr Ser Val Arg Phe Leu Gly Asp Gln Gln Arg Val Phe Leu Ser Lys     130 135 140 Asp Leu Leu Lys Pro Ile Gln Asp Val Asn Ser Leu Arg Leu Ser Leu 145 150 155 160 Thr Asp Asn Gln Ile Val Ser Lys Glu Phe Gln Ala Leu Ile Val Lys                 165 170 175 His Leu Asp Glu Ser His Leu Leu Lys Gly Asp Lys Asn Leu Val Gly             180 185 190 Ser Glu Val Lys Ile Tyr Ser Leu Asp Pro Ser Thr Gln Trp Phe Ser         195 200 205 Ala Thr Val Ile Asn Gly Asn Pro Ala Ser Lys Thr Leu Gln Val Asn     210 215 220 Cys Glu Glu Ile Pro Ala Leu Lys Ile Val Asp Pro Ser Leu Ile His 225 230 235 240 Val Glu Val Val His Asp Asn Leu Val Thr Cys Gly Asn Ser Ala Arg                 245 250 255 Ile Gly Ala Val Lys Arg Lys Ser Ser Glu Asn Asn Gly Thr Leu Val             260 265 270 Ser Lys Gln Ala Lys Ser Cys Ser Glu Ala Ser Pro Ser Met Cys Pro         275 280 285 Val Gln Ser Val Pro Thr Thr Val Phe Lys Glu Ile Leu Leu Gly Cys     290 295 300 Thr Ala Ala Thr Pro Pro Ser Lys Asp Pro Arg Gln Gln Ser Thr Pro 305 310 315 320 Gln Ala Ala Asn Ser Pro Pro Asn Leu Gly Ala Lys Ile Pro Gln Gly                 325 330 335 Cys His Lys Gln Ser Leu Pro Glu Glu Ile Ser Ser Cys Leu Asn Thr             340 345 350 Lys Ser Glu Ala Leu Arg Thr Lys Pro Asp Val Cys Lys Ala Gly Leu         355 360 365 Leu Ser Lys Ser Ser Gln Ile Gly Thr Gly Asp Leu Lys Ile Leu Thr     370 375 380 Glu Pro Lys Gly Ser Cys Thr Gln Pro Lys Thr Asn Thr Asp Gln Glu 385 390 395 400 Asn Arg Leu Glu Ser Val Pro Gln Ala Leu Thr Gly Leu Pro Lys Glu                 405 410 415 Cys Leu Pro Thr Lys Ala Ser Ser Lys Ala Glu Leu Glu Ile Ala Asn             420 425 430 Pro Pro Glu Leu Gln Lys His Leu Glu His Ala Pro Ser Pro Ser Asp         435 440 445 Val Ser Asn Ala Pro Glu Val Lys Ala Gly Val Asn Ser Asp Ser Pro     450 455 460 Asn Asn Cys Ser Gly Lys Lys Val Glu Pro Ser Ala Leu Ala Cys Arg 465 470 475 480 Ser Gln Asn Leu Lys Glu Ser Ser Val Lys Val Asp Asn Glu Ser Cys                 485 490 495 Cys Ser Arg Ser Asn Asn Lys Ile Gln Asn Ala Pro Ser Arg Lys Ser             500 505 510 Val Leu Thr Asp Pro Ala Lys Leu Lys Lys Leu Gln Gln Ser Gly Glu         515 520 525 Ala Phe Val Gln Asp Asp Ser Cys Val Asn Ile Val Ala Gln Leu Pro     530 535 540 Lys Cys Arg Glu Cys Arg Leu Asp Ser Leu Arg Lys Asp Lys Glu Gln 545 550 555 560 Gln Lys Asp Ser Pro Val Phe Cys Arg Phe Phe His Phe Arg Arg Leu                 565 570 575 Gln Phe Asn Lys His Gly Val Leu Arg Val Glu Gly Phe Leu Thr Pro             580 585 590 Asn Lys Tyr Asp Asn Glu Ala Ile Gly Leu Trp Leu Pro Leu Thr Lys         595 600 605 Asn Val Val Gly Ile Asp Leu Asp Thr Ala Lys Tyr Ile Leu Ala Asn     610 615 620 Ile Gly Asp His Phe Cys Gln Met Val Ile Ser Glu Lys Glu Ala Met 625 630 635 640 Ser Thr Ile Glu Pro His Arg Gln Val Ala Trp Lys Arg Ala Val Lys                 645 650 655 Gly Val Arg Glu Met Cys Asp Val Cys Asp Thr Thr Ile Phe Asn Leu             660 665 670 His Trp Val Cys Pro Arg Cys Gly Phe Gly Val Cys Val Asp Cys Tyr         675 680 685 Arg Met Lys Arg Lys Asn Cys Gln Gln Gly Ala Ala Tyr Lys Thr Phe     690 695 700 Ser Trp Leu Lys Cys Val Lys Ser Gln Ile His Glu Pro Glu Asn Leu 705 710 715 720 Met Pro Thr Gln Ile Ile Pro Gly Lys Ala Leu Tyr Asp Val Gly Asp                 725 730 735 Ile Val His Ser Val Arg Ala Lys Trp Gly Ile Lys Ala Asn Cys Pro             740 745 750 Cys Ser Asn Arg Gln Phe Lys Leu Phe Ser Lys Pro Ala Ser Lys Glu         755 760 765 Asp Leu Lys Gln Thr Ser Leu Ala Gly Glu Lys Pro Thr Leu Gly Ala     770 775 780 Val Leu Gln Gln Asn Pro Ser Val Leu Glu Pro Ala Ala Val Gly Gly 785 790 795 800 Glu Ala Ala Ser Lys Pro Ala Gly Ser Met Lys Pro Ala Cys Pro Ala                 805 810 815 Ser Thr Ser Pro Leu Asn Trp Leu Ala Asp Leu Thr Ser Gly Asn Val             820 825 830 Asn Lys Glu Asn Lys Glu Lys Gln Pro Thr Met Pro Ile Leu Lys Asn         835 840 845 Glu Ile Lys Cys Leu Pro Pro Leu Pro Pro Leu Ser Lys Ser Ser Thr     850 855 860 Val Leu His Thr Phe Asn Ser Thr Ile Leu Thr Pro Val Ser Asn Asn 865 870 875 880 Asn Ser Gly Phe Leu Arg Asn Leu Leu Asn Ser Ser Thr Gly Lys Thr                 885 890 895 Glu Asn Gly Leu Lys Asn Thr Pro Lys Ile Leu Asp Asp Ile Phe Ala             900 905 910 Ser Leu Val Gln Asn Lys Thr Thr Ser Asp Leu Ser Lys Arg Pro Gln         915 920 925 Gly Leu Thr Ile Lys Pro Ser Ile Leu Gly Phe Asp Thr Pro His Tyr     930 935 940 Trp Leu Cys Asp Asn Arg Leu Leu Cys Leu Gln Asp Pro Asn Asn Lys 945 950 955 960 Ser Asn Trp Asn Val Phe Arg Glu Cys Trp Lys Gln Gly Gln Pro Val                 965 970 975 Met Val Ser Gly Val His His Lys Leu Asn Ser Glu Leu Trp Lys Pro             980 985 990 Glu Ser Phe Arg Lys Glu Phe Gly Glu Gln Glu Val Asp Leu Val Asn         995 1000 1005 Cys Arg Thr Asn Glu Ile Ile Thr Gly Ala Thr Val Gly Asp Phe Trp    1010 1015 1020 Asp Gly Phe Glu Asp Val Pro Asn Arg Leu Lys Asn Glu Lys Glu Pro 1025 1030 1035 1040 Met Val Leu Lys Leu Lys Asp Trp Pro Pro Gly Glu Asp Phe Arg Asp                1045 1050 1055 Met Met Pro Ser Arg Phe Asp Asp Leu Met Ala Asn Ile Pro Leu Pro            1060 1065 1070 Glu Tyr Thr Arg Arg Asp Gly Lys Leu Asn Leu Ala Ser Arg Leu Pro        1075 1080 1085 Asn Tyr Phe Val Arg Pro Asp Leu Gly Pro Lys Met Tyr Asn Ala Tyr    1090 1095 1100 Gly Leu Ile Thr Pro Glu Asp Arg Lys Tyr Gly Thr Thr Asn Leu His 1105 1110 1115 1120 Leu Asp Val Ser Asp Ala Ala Asn Val Met Val Tyr Val Gly Ile Pro                1125 1130 1135 Lys Gly Gln Cys Glu Gln Glu Glu Glu Val Leu Lys Thr Ile Gln Asp            1140 1145 1150 Gly Asp Ser Asp Glu Leu Thr Ile Lys Arg Phe Ile Glu Gly Lys Glu        1155 1160 1165 Lys Pro Gly Ala Leu Trp His Ile Tyr Ala Ala Lys Asp Thr Glu Lys    1170 1175 1180 Ile Arg Glu Phe Leu Lys Lys Val Ser Glu Glu Gln Gly Gln Glu Asn 1185 1190 1195 1200 Pro Ala Asp His Asp Pro Ile His Asp Gln Ser Trp Tyr Leu Asp Arg                1205 1210 1215 Ser Leu Arg Lys Arg Leu His Gln Glu Tyr Gly Val Gln Gly Trp Ala            1220 1225 1230 Ile Val Gln Phe Leu Gly Asp Val Val Phe Ile Pro Ala Gly Ala Pro        1235 1240 1245 His Gln Val His Asn Leu Tyr Ser Cys Ile Lys Val Ala Glu Asp Phe    1250 1255 1260 Val Ser Pro Glu His Val Lys His Cys Phe Trp Leu Thr Gln Glu Phe 1265 1270 1275 1280 Arg Tyr Leu Ser Gln Thr His Thr Asn His Glu Asp Lys Leu Gln Val                1285 1290 1295 Lys Asn Val Ile Tyr His Ala Val Lys Asp Ala Val Ala Met Leu Lys            1300 1305 1310 Ala Ser Glu Ser Ser Phe Gly Lys Pro        1315 1320 <210> 2 <211> 1132 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <222> (1) .. (1132) <223> JAK2 <400> 2 Met Gly Met Ala Cys Leu Thr Met Thr Glu Met Glu Gly Thr Ser Thr   1 5 10 15 Ser Ser Ile Tyr Gln Asn Gly Asp Ile Ser Gly Asn Ala Asn Ser Met              20 25 30 Lys Gln Ile Asp Pro Val Leu Gln Val Tyr Leu Tyr His Ser Leu Gly          35 40 45 Lys Ser Glu Ala Asp Tyr Leu Thr Phe Pro Ser Gly Glu Tyr Val Ala      50 55 60 Glu Glu Ile Cys Ile Ala Ala Ser Lys Ala Cys Gly Ile Thr Pro Val  65 70 75 80 Tyr His Asn Met Phe Ala Leu Met Ser Glu Thr Glu Arg Ile Trp Tyr                  85 90 95 Pro Pro Asn His Val Phe His Ile Asp Glu Ser Thr Arg His Asn Val             100 105 110 Leu Tyr Arg Ile Arg Phe Tyr Phe Pro Arg Trp Tyr Cys Ser Gly Ser         115 120 125 Asn Arg Ala Tyr Arg His Gly Ile Ser Arg Gly Ala Glu Ala Pro Leu     130 135 140 Leu Asp Asp Phe Val Met Ser Tyr Leu Phe Ala Gln Trp Arg His Asp 145 150 155 160 Phe Val His Gly Trp Ile Lys Val Pro Val Thr His Glu Thr Gln Glu                 165 170 175 Glu Cys Leu Gly Met Ala Val Leu Asp Met Met Arg Ile Ala Lys Glu             180 185 190 Asn Asp Gln Thr Pro Leu Ala Ile Tyr Asn Ser Ile Ser Tyr Lys Thr         195 200 205 Phe Leu Pro Lys Cys Ile Arg Ala Lys Ile Gln Asp Tyr His Ile Leu     210 215 220 Thr Arg Lys Arg Ile Arg Tyr Arg Phe Arg Arg Phe Ile Gln Gln Phe 225 230 235 240 Ser Gln Cys Lys Ala Thr Ala Arg Asn Leu Lys Leu Lys Tyr Leu Ile                 245 250 255 Asn Leu Glu Thr Leu Gln Ser Ala Phe Tyr Thr Glu Lys Phe Glu Val             260 265 270 Lys Glu Pro Gly Ser Gly Pro Ser Gly Glu Glu Ile Phe Ala Thr Ile         275 280 285 Ile Ile Thr Gly Asn Gly Gly Ile Gln Trp Ser Arg Gly Lys His Lys     290 295 300 Glu Ser Glu Thr Leu Thr Glu Gln Asp Leu Gln Leu Tyr Cys Asp Phe 305 310 315 320 Pro Asn Ile Ile Asp Val Ser Ile Lys Gln Ala Asn Gln Glu Gly Ser                 325 330 335 Asn Glu Ser Arg Val Val Thr Ile His Lys Gln Asp Gly Lys Asn Leu             340 345 350 Glu Ile Glu Leu Ser Ser Leu Arg Glu Ala Leu Ser Phe Val Ser Leu         355 360 365 Ile Asp Gly Tyr Tyr Arg Leu Thr Ala Asp Ala His His Tyr Leu Cys     370 375 380 Lys Glu Val Ala Pro Pro Ala Val Leu Glu Asn Ile Gln Ser Asn Cys 385 390 395 400 His Gly Pro Ile Ser Met Asp Phe Ala Ile Ser Lys Leu Lys Lys Ala                 405 410 415 Gly Asn Gln Thr Gly Leu Tyr Val Leu Arg Cys Ser Pro Lys Asp Phe             420 425 430 Asn Lys Tyr Phe Leu Thr Phe Ala Val Glu Arg Glu Asn Val Ile Glu         435 440 445 Tyr Lys His Cys Leu Ile Thr Lys Asn Glu Asn Glu Glu Tyr Asn Leu     450 455 460 Ser Gly Thr Lys Lys Asn Phe Ser Ser Leu Lys Asp Leu Leu Asn Cys 465 470 475 480 Tyr Gln Met Glu Thr Val Arg Ser Asp Asn Ile Ile Phe Gln Phe Thr                 485 490 495 Lys Cys Cys Pro Pro Lys Pro Lys Asp Lys Ser Asn Leu Leu Val Phe             500 505 510 Arg Thr Asn Gly Val Ser Asp Val Pro Thr Ser Pro Thr Leu Gln Arg         515 520 525 Pro Thr His Met Asn Gln Met Val Phe His Lys Ile Arg Asn Glu Asp     530 535 540 Leu Ile Phe Asn Glu Ser Leu Gly Gln Gly Thr Phe Thr Lys Ile Phe 545 550 555 560 Lys Gly Val Arg Arg Glu Val Gly Asp Tyr Gly Gln Leu His Glu Thr                 565 570 575 Glu Val Leu Leu Lys Val Leu Asp Lys Ala His Arg Asn Tyr Ser Glu             580 585 590 Ser Phe Phe Glu Ala Ala Ser Met Met Ser Lys Leu Ser His Lys His         595 600 605 Leu Val Leu Asn Tyr Gly Val Cys Val Cys Gly Asp Glu Asn Ile Leu     610 615 620 Val Gln Glu Phe Val Lys Phe Gly Ser Leu Asp Thr Tyr Leu Lys Lys 625 630 635 640 Asn Lys Asn Cys Ile Asn Ile Leu Trp Lys Leu Glu Val Ala Lys Gln                 645 650 655 Leu Ala Trp Ala Met His Phe Leu Glu Glu Asn Thr Leu Ile His Gly             660 665 670 Asn Val Cys Ala Lys Asn Ile Leu Leu Ile Arg Glu Glu Asp Arg Lys         675 680 685 Thr Gly Asn Pro Pro Phe Ile Lys Leu Ser Asp Pro Gly Ile Ser Ile     690 695 700 Thr Val Leu Pro Lys Asp Ile Leu Gln Glu Arg Ile Pro Trp Val Pro 705 710 715 720 Pro Glu Cys Ile Glu Asn Pro Lys Asn Leu Asn Leu Ala Thr Asp Lys                 725 730 735 Trp Ser Phe Gly Thr Thr Leu Trp Glu Ile Cys Ser Gly Gly Asp Lys             740 745 750 Pro Leu Ser Ala Leu Asp Ser Gln Arg Lys Leu Gln Phe Tyr Glu Asp         755 760 765 Arg His Gln Leu Pro Ala Pro Lys Trp Ala Glu Leu Ala Asn Leu Ile     770 775 780 Asn Asn Cys Met Asp Tyr Glu Pro Asp Phe Arg Pro Ser Phe Arg Ala 785 790 795 800 Ile Ile Arg Asp Leu Asn Ser Leu Phe Thr Pro Asp Tyr Glu Leu Leu                 805 810 815 Thr Glu Asn Asp Met Leu Pro Asn Met Arg Ile Gly Ala Leu Gly Phe             820 825 830 Ser Gly Ala Phe Glu Asp Arg Asp Pro Thr Gln Phe Glu Glu Arg His         835 840 845 Leu Lys Phe Leu Gln Gln Leu Gly Lys Gly Asn Phe Gly Ser Val Glu     850 855 860 Met Cys Arg Tyr Asp Pro Leu Gln Asp Asn Thr Gly Glu Val Val Ala 865 870 875 880 Val Lys Lys Leu Gln His Ser Thr Glu Glu His Leu Arg Asp Phe Glu                 885 890 895 Arg Glu Ile Glu Ile Leu Lys Ser Leu Gln His Asp Asn Ile Val Lys             900 905 910 Tyr Lys Gly Val Cys Tyr Ser Ala Gly Arg Arg Asn Leu Lys Leu Ile         915 920 925 Met Glu Tyr Leu Pro Tyr Gly Ser Leu Arg Asp Tyr Leu Gln Lys His     930 935 940 Lys Glu Arg Ile Asp His Ile Lys Leu Leu Gln Tyr Thr Ser Gln Ile 945 950 955 960 Cys Lys Gly Met Glu Tyr Leu Gly Thr Lys Arg Tyr Ile His Arg Asp                 965 970 975 Leu Ala Thr Arg Asn Ile Leu Val Glu Asn Glu Asn Arg Val Lys Ile             980 985 990 Gly Asp Phe Gly Leu Thr Lys Val Leu Pro Gln Asp Lys Glu Tyr Tyr         995 1000 1005 Lys Val Lys Glu Pro Gly Glu Ser Pro Ile Phe Trp Tyr Ala Pro Glu    1010 1015 1020 Ser Leu Thr Glu Ser Lys Phe Ser Val Ala Ser Asp Val Trp Ser Phe 1025 1030 1035 1040 Gly Val Val Leu Tyr Glu Leu Phe Thr Tyr Ile Glu Lys Ser Lys Ser                1045 1050 1055 Pro Pro Ala Glu Phe Met Arg Met Ile Gly Asn Asp Lys Gln Gly Gln            1060 1065 1070 Met Ile Val Phe His Leu Ile Glu Leu Leu Lys Asn Asn Gly Arg Leu        1075 1080 1085 Pro Arg Pro Asp Gly Cys Pro Asp Glu Ile Tyr Met Ile Met Thr Glu    1090 1095 1100 Cys Trp Asn Asn Asn Val Asn Gln Arg Pro Ser Phe Arg Asp Leu Ala 1105 1110 1115 1120 Leu Arg Val Asp Gln Ile Arg Asp Asn Met Ala Gly                1125 1130 <210> 3 <211> 770 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <222> (1) .. (770) <223> STAT3 <400> 3 Met Ala Gln Trp Asn Gln Leu Gln Gln Leu Asp Thr Arg Tyr Leu Glu   1 5 10 15 Gln Leu His Gln Leu Tyr Ser Asp Ser Phe Pro Met Glu Leu Arg Gln              20 25 30 Phe Leu Ala Pro Trp Ile Glu Ser Gln Asp Trp Ala Tyr Ala Ala Ser          35 40 45 Lys Glu Ser His Ala Thr Leu Val Phe His Asn Leu Leu Gly Glu Ile      50 55 60 Asp Gln Gln Tyr Ser Arg Phe Leu Gln Glu Ser Asn Val Leu Tyr Gln  65 70 75 80 His Asn Leu Arg Arg Ile Lys Gln Phe Leu Gln Ser Arg Tyr Leu Glu                  85 90 95 Lys Pro Met Glu Ile Ala Arg Ile Val Ala Arg Cys Leu Trp Glu Glu             100 105 110 Ser Arg Leu Leu Gln Thr Ala Ala Thr Ala Ala Gln Gln Gly Gly Gln         115 120 125 Ala Asn His Pro Thr Ala Ala Val Val Thr Glu Lys Gln Gln Met Leu     130 135 140 Glu Gln His Leu Gln Asp Val Arg Lys Arg Val Gln Asp Leu Glu Gln 145 150 155 160 Lys Met Lys Val Val Glu Asn Leu Gln Asp Asp Phe Asp Phe Asn Tyr                 165 170 175 Lys Thr Leu Lys Ser Gln Gly Asp Met Gln Asp Leu Asn Gly Asn Asn             180 185 190 Gln Ser Val Thr Arg Gln Lys Met Gln Gln Leu Glu Gln Met Leu Thr         195 200 205 Ala Leu Asp Gln Met Arg Arg Ser Ile Val Ser Glu Leu Ala Gly Leu     210 215 220 Leu Ser Ala Met Glu Tyr Val Gln Lys Thr Leu Thr Asp Glu Glu Leu 225 230 235 240 Ala Asp Trp Lys Arg Arg Gln Gln Ile Ala Cys Ile Gly Gly Pro Pro                 245 250 255 Asn Ile Cys Leu Asp Arg Leu Glu Asn Trp Ile Thr Ser Leu Ala Glu             260 265 270 Ser Gln Leu Gln Thr Arg Gln Gln Ile Lys Lys Leu Glu Glu Leu Gln         275 280 285 Gln Lys Val Ser Tyr Lys Gly Asp Pro Ile Val Gln His Arg Pro Met     290 295 300 Leu Glu Glu Arg Ile Val Glu Leu Phe Arg Asn Leu Met Lys Ser Ala 305 310 315 320 Phe Val Val Glu Arg Gln Pro Cys Met Pro Met His Pro Asp Arg Pro                 325 330 335 Leu Val Ile Lys Thr Gly Val Gln Phe Thr Thr Lys Val Arg Leu Leu             340 345 350 Val Lys Phe Pro Glu Leu Asn Tyr Gln Leu Lys Ile Lys Val Cys Ile         355 360 365 Asp Lys Asp Ser Gly Asp Val Ala Ala Leu Arg Gly Ser Arg Lys Phe     370 375 380 Asn Ile Leu Gly Thr Asn Thr Lys Val Met Asn Met Glu Glu Ser Asn 385 390 395 400 Asn Gly Ser Leu Ser Ala Glu Phe Lys His Leu Thr Leu Arg Glu Gln                 405 410 415 Arg Cys Gly Asn Gly Gly Arg Ala Asn Cys Asp Ala Ser Leu Ile Val             420 425 430 Thr Glu Glu Leu His Leu Ile Thr Phe Glu Thr Glu Val Tyr His Gln         435 440 445 Gly Leu Lys Ile Asp Leu Glu Thr His Ser Leu Pro Val Val Val Ile     450 455 460 Ser Asn Ile Cys Gln Met Pro Asn Ala Trp Ala Ser Ile Leu Trp Tyr 465 470 475 480 Asn Met Leu Thr Asn Asn Pro Lys Asn Val Asn Phe Phe Thr Lys Pro                 485 490 495 Pro Ile Gly Thr Trp Asp Gln Val Ala Glu Val Leu Ser Trp Gln Phe             500 505 510 Ser Ser Thr Thr Lys Arg Gly Leu Ser Ile Glu Gln Leu Thr Thr Leu         515 520 525 Ala Glu Lys Leu Leu Gly Pro Gly Val Asn Tyr Ser Gly Cys Gln Ile     530 535 540 Thr Trp Ala Lys Phe Cys Lys Glu Asn Met Ala Gly Lys Gly Phe Ser 545 550 555 560 Phe Trp Val Trp Leu Asp Asn Ile Ile Asp Leu Val Lys Lys Tyr Ile                 565 570 575 Leu Ala Leu Trp Asn Glu Gly Tyr Ile Met Gly Phe Ile Ser Lys Glu             580 585 590 Arg Glu Arg Ala Ile Leu Ser Thr Lys Pro Pro Gly Thr Phe Leu Leu         595 600 605 Arg Phe Ser Glu Ser Ser Lys Glu Gly Gly Val Thr Phe Thr Trp Val     610 615 620 Glu Lys Asp Ile Ser Gly Lys Thr Gln Ile Gln Ser Val Glu Pro Tyr 625 630 635 640 Thr Lys Gln Gln Leu Asn Asn Met Ser Phe Ala Glu Ile Ile Met Gly                 645 650 655 Tyr Lys Ile Met Asp Ala Thr Asn Ile Leu Val Ser Pro Leu Val Tyr             660 665 670 Leu Tyr Pro Asp Ile Pro Lys Glu Glu Ala Phe Gly Lys Tyr Cys Arg         675 680 685 Pro Glu Ser Gln Glu His Pro Glu Ala Asp Pro Gly Ser Ala Ala Pro     690 695 700 Tyr Leu Lys Thr Lys Phe Ile Cys Val Thr Pro Thr Thr Cys Ser Asn 705 710 715 720 Thr Ile Asp Leu Pro Met Ser Pro Arg Thr Leu Asp Ser Leu Met Gln                 725 730 735 Phe Gly Asn Asn Gly Glu Gly Ala Glu Pro Ser Ala Gly Gly Gln Phe             740 745 750 Glu Ser Leu Thr Phe Asp Met Glu Leu Thr Ser Glu Cys Ala Thr Ser         755 760 765 Pro Met     770 <210> 4 <211> 135 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <222> (1) .. (135) <223> HISTONE 3 <400> 4 Ala Arg Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys Ala Pro   1 5 10 15 Arg Lys Gln Leu Ala Thr Lys Val Ala Arg Lys Ser Ala Pro Ala Thr              20 25 30 Gly Gly Val Lys Lys Pro His Arg Tyr Arg Pro Gly Thr Val Ala Leu          35 40 45 Arg Glu Ile Arg Arg Tyr Gln Lys Ser Thr Glu Leu Leu Ile Arg Lys      50 55 60 Leu Pro Phe Gln Arg Leu Met Arg Glu Ile Ala Gln Asp Phe Lys Thr  65 70 75 80 Asp Leu Arg Phe Gln Ser Ser Ala Val Met Ala Leu Gln Glu Ala Cys                  85 90 95 Glu Ser Tyr Leu Val Gly Leu Phe Glu Asp Thr Asn Leu Cys Val Ile             100 105 110 His Ala Lys Arg Val Thr Ile Met Pro Lys Asp Ile Gln Leu Ala Arg         115 120 125 Arg Ile Arg Gly Glu Arg Ala     130 135

Claims (10)

A screening method for a substance for treating cancer related to abnormal signal transducer and activator of Transcription 3 (JAK2) -STAT3 (January tyrosine kinase 2) -STAT3 via lysine-specific demethylase 3A (KDM3A), the method comprising
Among the presence of a test substance expected to inhibit phosphorylation of the tyrosine residue by JAK2, the KDM3A protein comprising the 1101 th tyrosine residue based on the JAK2 protein and the sequence of SEQ ID NO: 1 that can be phosphorylated by the JAK2 protein. A first step of culturing;
A second step of measuring the phosphorylation degree of the tyrosine residue of the KDM3A protein; And
As a result of the measurement, when the phosphorylation of the KDM3A protein is reduced compared to the control group not treated with the test substance, the third step of selecting it as a candidate substance capable of inhibiting the growth of cancer cells , JAK2-STAT3 signaling method for the treatment of cancer by abnormal signaling.
According to claim 1,
The first step further comprises a STAT3 protein,
In the second step, further comprising measuring the activity of the KDM3A protein as a cofactor for the STAT2 protein,
In the above step 3, when the measurement result is compared to the control group not treated with the test substance, when treated with the test substance, the activity of the KDM3A protein as a cofactor for the STAT3 protein, when the transcriptional activity of the STAT3 protein is reduced , Further comprising selecting it as a candidate for inhibiting the growth of cancer cells.
The method of claim 1 or 2,
The first step further comprises a histone 3 protein (H3K9, Histone 3 Lysine 9) methylated with a ninth lysine residue based on SEQ ID NO: 4,
In the second step, further comprising measuring the demethylation activity of the KDM3A protein for H3K9,
Compared to the control group not treated with the test substance in the measurement step in step 3, when the demethylation of the H3K9 protein is reduced when treated with the test substance, it is further selected as a candidate substance that inhibits the growth of cancer cells. Included with, method.
The method according to any one of claims 1 to 3,
The first and second steps are performed in eukaryotic cells expressing the respective proteins, and the cells are further treated with a substance capable of activating the JAK2-STAT signaling pathway.
The method of claim 4,
The substance capable of activating the JAK2-STAT signaling pathway is a signal transduction molecule mediated by gp (glycoprotein) 130 including IL (Interleukin) -6, IL-11 or Oncostatin M (OSM).
The method according to any one of claims 1 to 5,
The cancer is a solid cancer, including cervical cancer, ovarian cancer, breast cancer, kidney cancer and stomach cancer.
An antibody or antigen-binding fragment thereof that specifically recognizes KDM3A phosphorylated with the 1101 th tyrosine residue based on the sequence of SEQ ID NO: 1.
An antibody or antigen-binding fragment thereof that specifically recognizes the KMpYNAYGLI epitope to which the KDM3A protein 1101 th tyrosine residue is phosphorylated based on the sequence of SEQ ID NO: 1.
The method of claim 7 or 8,
The antibody is a polyclonal or monoclonal, antibody or antigen-binding fragment.
The method of claim 7 or 8,
The antigen-binding fragment is Fv, ScFv, Fab, Fab 'and F (ab') 2, antibody or antigen-binding fragment.

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