KR102108414B1 - Use for regulating cancer cell differentiation by regulating AURKA-mediated KDM6B activity - Google Patents

Abstract

The present invention relates to use for regulating AURKA-mediated KDM6B signaling activity during a leukemia cell differentiation process. More specifically, in PMA-treated THP-1 cells, AURKA was found to collect YY1 with a KDM6B promoter, interact with the collected YY1, and regulate the expression of KDM6B and KDM6B-dependent p21 by binding to the promoter of KDM6B through the interacted YY1, thereby inhibiting THP-1 cell differentiation. Therefore, the AURKA-KDM6B signaling process or binding regulation can induce the differentiation of leukemia cells so that the present invention can be provided as a novel anticancer drug development strategy and anticancer drug screening method.

Description

Use for regulating cancer cell differentiation by regulating AURKA-mediated KDM6B activity}

It relates to a leukemia cell differentiation control through AURKA-mediated KDM6B signal activity regulation during leukemia cell differentiation process.

Mixed-lineage leukemia (MLL) proteins are fused with over 60 partners ranging from transcription factors to cytoplasmic structural proteins.

AF9 is the most common fusion partner, found in 30% of MLL-rearranged acute myeloid leukemia (AML), and MLL-AF9 leukemia, which accounts for 10% of acute myeloid cases, is usually prognostic Is not good

THP-1 cells, widely used for MLL-AF9-driven AML studies, are acute monocyte leukemia cell lines that are similar to primary human monocytes and are well known to differentiate into macrophages by external stimulation. Common MLL fusion proteins, including MLL-AF4, MLL-AF9, MLL-ENL, and MLL-ELL, cause leukemia through dysregulation of MLL target gene expression, thus inhibiting MLL fusion oncogene-engineered proteins, including DOT1L and BRD4 inhibitors Can be used as a potential therapeutic.

A recent study report confirmed that ATRA induces granulocyte differentiation in acute promyelocytic leukemia. Differentiation therapy is little known about the treatment method of treating differentiated cells by inducing the final differentiation of malignant cells or the differentiation mechanism of MLL fusion protein-induced leukemia THP-1 cells.

Aurora kinase A (AURKA) is a serine / threonine protein kinase that is amplified in many malignant tumors, including AML.The AURK group induces mitotic phosphorylation of histone H3 serine 10 (H3S10) in germinating yeast and nematodes, In particular, it exhibits excellent kinase activity against H3S10. Notably, H3S10 phosphorylation is associated with mitotic chromatin concentration in the early G2 stage as well as cell cycle progression. Recent studies have reported that AURKA can regulate the level of transcription of a target gene through catalytic activity.

Lysine (K) demethylase 6 (KDM6) group protein has histone H3 lysine 27 (H3K27) demethylation activity, and KDM6A and KDM6B are both well-known tumor markers for MLL rearranged leukemia, the HOX gene. It has tumor suppressing properties such as regulation. According to previous research reports, KDM6A is easily targeted in several types of MLL fusion leukemia cell lines with somatic mutation, and KDM6B has been confirmed to stop the cell cycle by upregulating the cell cycle inhibitor p21.

However, the mechanism of AURKA-KDM6B signaling in the treatment of leukemia has not been clearly identified.

U.S. Patent Publication No. 2012-0070450 (2012.03.22. Published)

An object of the present invention is to provide a method for screening for a leukemia therapeutic agent, comprising the step of selecting a test substance having reduced AURKA and YY1 binding levels.

In addition, it contains AURKA as an active ingredient, the AURKA is a reagent composition for inhibiting the expression of a protein selected from the group consisting of KDM6B and p21 of leukemia cells in vitro and a protein selected from the group consisting of KDM6B and p21 of leukemia cells. It is intended to provide a method for inhibiting the expression of.

The present invention comprises the steps of (1) treating a test substance on leukemia cells; (2) confirming the binding level of AURKA (Aurora kinase A) and YY1 in leukemia cells treated with the test substance; And (3) provides a method for screening for a leukemia therapeutic agent, comprising selecting a test substance that reduces the AURKA and YY1 binding level compared to a control sample.

In addition, the present invention contains AURKA as an active ingredient, and AURKA provides a reagent composition for inhibiting the expression of a protein selected from the group consisting of KDM6B and p21 of leukemia cells in vitro.

In addition, the present invention provides a method for inhibiting the expression of a protein selected from the group consisting of KDM6B and p21 of leukemia cells comprising the step of treating AURKA with leukemia cells in vitro.

According to the present invention, in PMA-treated THP-1 cells, AURKA recruited YY1 with the KDM6B promoter and interacted with the recruited YY1, and by binding to the promoter of KDM6B through the interacted YY1, KDM6B and KDM6B dependence p21 As it is confirmed to inhibit THP-1 cell differentiation by regulating the expression of AURKA-KDM6B signaling process or binding regulation can induce differentiation of leukemia cells, it can be provided as a novel anticancer drug development strategy and anticancer drug screening method have.

1 is a result of confirming the level of H3S10 phosphorylation reduction during leukemia cell differentiation, and FIG. 1 (A) is culturing THP-1 cells for 48 hours in the presence of 100 ng / ml PMA or DMSO and CD14 and CD11b confirmed by qRT-PCR The expression level is normalized to GAPDH, and the result is expressed as mean ± SEM (n = 3; * p <0.05, ** p <0.01.), And FIG. 1 (B) is treated with 100 ng / ml PMA for 48 hours. As a result of Western blot analysis confirming the level of histone modification during the THP-1 differentiation, H3 was used as a loading control, and FIG. 1 (C) confirmed the AURKA and AURKB expression levels during the THP-1 differentiation by qRT-PCR and GAPDH Normalized to the mean ± SEM (n = 3; * p <0.05, ** p <0.01.) Is the result, Figure 1 (D) is 100 ng / ml PMA in THMA-1 cells grown for 48 hours and After dissolving, Western blot was performed using an anti-AURKA antibody, GAPDH was used as a loading control, and FIG. 1 (E) stably used AURKA. CD14 and CD11b expression levels in knocked down THP-1 cells were confirmed by qRT-PCR and normalized to GAPDH, resulting in mean ± SEM (n = 3; * p <0.05, ** p <0.01), and FIG. 1 ( F) The left figure shows the mean ± SEM (n = 3; * p <0.05, ** normalized to GAPDH by confirming the CD14 and CD11b expression levels in qP-PCR in allertip or DMSO treated THP-1 cells for 48 hours. p <0.01), and the figure on the right shows the histone modification level by performing Western blot using each antibody, and the result using H3 as a loading control. All experiments were repeated three times, showing similar results.
Figure 2 is a result of confirming the transcription control and p21 expression of KDM6B in PMA-treated THP-1 cells, Figure 2 (A) is treated with 100 ng / ml PMA to induce differentiation of THP-1 cells and KDM6B after 48 hours , RFX2, CDK19, CPT1A and SFRP1 expression levels were confirmed by qRT-PCR and normalized by GAPDH, and the results are shown as mean ± SEM (n = 3; * p <0.05, ** p <0.01), and FIG. 2 (B) The result was expressed as mean ± SEM (n = 3; *** p <0.001) by treating allertip or DMSO in THP-1 cells for 48 hours and confirming KDM6B expression level using qRT-PCR, then normalizing to GAPDH. Figure 2 (C), HEK 293T cells were transfected with a pGL3KDM6B promoter reporter DNA construct, and after treatment with allertip or DMSO to the cells, the cell extract was analyzed for luciferase activity, and the activity was β- It was normalized for galactosidase, and is a result represented by mean ± SEM (n = 4; * p <0.05), and FIG. 2 (D) shows HEK 293T As a result of analyzing the luciferase activity in the cell extract after transfection of the cells with the pGL3KDM6B promoter reporter and AURKA DNA construct, β-galactosidase was used for normalization and the mean ± SEM, (n = 4; * p < 0.05, *** p <0.001), and FIG. 2 (E) shows the expression level of p21 by growing THP-1 cells with allertip or DMSO and performing qRT-PCR and Western blotting after 48 hours. As a result of comparison, GAPDH was used as a control, and results are shown as mean ± SEM (n = 3; ** p <0.01.), And FIG. 2 (F) shows the pGL3p21 promoter reporter and AURKA DNA construct in HEK 293T cells. As a result of confirming the luciferase activity in the cell extract after transfection, the luciferase activity was normalized to β-galactosidase and then averaged ± SEM (n = 4; ** p <0.01, *** p <0.001), and FIG. 2 (G) shows ectopic expression of the pGL3p21 promoter reporter in HEK 293T cells, followed by aliser tip (0.1 μM or 0.3 μM) or DMSO in the cells. After 24 hours of treatment, cells were lysed to confirm luciferase activity. After normalizing with β-galactosidase, the results are shown as mean ± SEM (n = 4; * p <0.05). All experiments were repeated three times, showing similar results.
3 is a result of confirming that AURKA inhibits the expression of KDM6B through recruitment of YY1, and FIG. 3 (A) is a schematic diagram of predicting YY1 and GATA1 binding sites in the KDM6B promoter region. It is a result of predicting the YY1 binding site, and FIG. 3 (B) shows the immunoprecipitation and related proteins using anti-Flag and anti-GFP antibodies after lysis of HEK 293T cells transfected with GFP-AURKA and Flag-YY1. After eluting, it was decomposed on SDS-PAGE and immunoblotted using each antibody. FIG. 3 (C) shows cell-lysis of HEK 293T cells transfected with GFP-AURKA and Flag-YY1, followed by anti-Flag. And immunoprecipitation using an anti-GFP antibody, and dissociation in a related SDS-PAGE where the related protein was eluted, and immunoblotting using an antibody, respectively. FIG. 3 (D) shows the pGL3KDM6B promoter reporter and AURKA and YY1 DNA constructs. HEK 293T transfected with The luciferase activity was confirmed in the extract of Po, normalized to β-galactosidase, and the results are shown as mean ± SEM (n = 4; * p <0.05, ** p <0.01), and FIG. 3 (E) shows the pGL3KDM6B promoter reporter. And luciferase activity in cell lysates of HEK 293T cells transfected with AURKA and GATA1 DNA constructs, normalized to β-galactosidase, and expressed as mean ± SEM (n = 4; ** p <0.01) 3 (F) was treated with 100 ng / ml PMA to induce differentiation of THP-1 cells, and after 48 hours, ChIP analysis was performed using anti-IgG, anti-AURKA, and anti-YY1 antibodies. RT-PCR is performed to analyze the immunoprecipitated DNA fragments from the proximal region of KDM6B, and the results are shown as mean ± SD, and FIG. 3 (G) shows anti-IgG after treatment with 0.3 μM of allertip for 24 hours. And ChIP analysis was performed with an anti-YY1 antibody, and RT-PCR was performed to attach to the proximal region of KDM6B. The results of the analysis of the immunoprecipitated DNA fragments were expressed as mean ± SD. All experiments were repeated three times, showing similar results.
4 is a result of confirming p21 expression regulated by KDM6B during the PMA-mediated THP-1 differentiation period, and FIG. 4 (A) is culturing THP-1 cells with 100 ng / ml PMA, 5 μM GSK-J4 or DMSO After 48 hours, qRT-PCR was performed to compare the expression level of the p21 gene and normalize it to GAPDH, and the results are shown as mean ± SEM (n = 3; ** p <0.01), and FIG. 4 (B) Is a result of analyzing the luciferase activity in the cell extract of HEK 293T cells transfected with the pGL3p21 promoter reporter and KDM6B DNA construct, normalized with β-galactosidase, and expressed as mean ± SEM (n = 4; * p <0.05) Figure 4 (C) was treated with GSK-J4 (2 μM or 5 μM) or DMSO in HEK 293T cells expressing ectopic pGL3p21 promoter reporter and lysed the cells after 24 hours to confirm luciferase activity, β-gal Normalized with lactosidase, the results are expressed as mean ± SEM (n = 4; * p <0.01), and FIG. HEK 293T cells overexpressing the L3p21 promoter reporter were treated with 0.3 μM allertip, 2 or 5 μM GSK-J4, or DMSO and lysed the cells to confirm luciferase activity, normalized with β-galactosidase, and averaged ± SEM (n = 4; * p <0.05, ** p <0.01), and FIG. 4 (E) shows anti-H3K27me3, anti-H3K4me3 and anti-IgG antibodies in allertip treated THP-1 cells. As a result of performing ChIP analysis of the p21 promoter and qRT-PCR analysis, recruitment of each histone modification to the promoter region was normalized and expressed as a% Input value, and the result value was expressed as a mean ± SD of a technical triple. As a result, all experiments were repeated three times, showing similar results.
Figure 5 is a result of confirming the acceleration of THP-1 cell differentiation by KDM6B, Figure 5 (A) is qRT in THP-1 cells treated with 100 ng / ml PMA, 2 μM GSK-J4, or DMSO for 48 hours -As a result of confirming the CD14 and CD11b expression levels by performing PCR and normalizing with GAPD, the values are shown as mean ± SEM (n = 3; * p <0.05, ** p <0.01), and FIG. 5 (B ) Is a negative control (shNC) and shKDM6B transfected THP-1 cells treated with 100 ng / ml PMA for 48 hours, staining the cells with PE-CD11b and APC-CD14 antibodies and showing the percentage of cells in each quadrant. Figure 5 (C) is a result showing the percentage of cells in each quadrant after staining with PE-CD11b and APC-CD14 antibody after treating 2 μM GSK-J4 or 0.3 μM allertip with THP-1 cells for 48 hours. , FIG. 5 (D) is a schematic diagram showing an AURKA model that regulates KDM6B expression in PMA-mediated THP-1 differentiation.

Hereinafter, the present invention will be described in more detail.

While studying the differentiation process of leukemia cells stagnant in the G0 / G1 cell cycle, the present inventors confirmed that while AURKA expression was decreased during THP-1 cell differentiation, KDM6B and KDM6B-dependent p21 expression was increased and THP-1 As a result of confirming the AURKA-KDM6B signaling process to confirm the role of AURKA in cell differentiation, AURKA recruited YY1 as a KDM6B promoter and interacted with the recruited YY1 in THP-1 cells treated with PMA. While binding to the promoter of KDM6B through YY1 acted to suppress the differentiation of THP-1 cells by regulating the expression of KDM6B and KDM6B-dependent p21, when the activity or expression of AURKA is decreased, the KDM6B promoter region in THP-1 cells It was found that YY1 was isolated from KD6B and increased expression of KDM6B-dependent p21 to induce differentiation of THP-1 cells into monocytes, and completed the present invention.

The present invention comprises the steps of (1) treating a test substance on leukemia cells; (2) confirming the binding level of AURKA (Aurora kinase A) and YY1 in leukemia cells treated with the test substance; And (3) a screening method for a leukemia therapeutic agent, comprising selecting a test substance that reduces the AURKA and YY1 binding level compared to a control sample.

The AURKA may be bound to YY1, and YY1 bound to AURKA may bind to the transcriptional regulatory site of KDM6B.

The AURKA and YY1 binding may be to inhibit the differentiation of leukemia cells by inhibiting KDM6B-mediated p21 expression by inducing KDM6B expression inhibition.

The AURKA and YY1 binding level may be measured by immunoprecipitation or immunoblotting.

The term "test substance" used while referring to the screening method of the present invention is used to test whether it affects the expression level of a gene, the expression or activity of a protein, or the binding between proteins. Refers to an unknown candidate substance used in screening. The sample includes, but is not limited to, chemicals, nucleotides, antisense-RNA, small interference RNA (siRNA), and natural product extracts.

In addition, the present invention contains AURKA as an active ingredient, the AURKA can provide a reagent composition for inhibiting the expression of a protein selected from the group consisting of KDM6B and p21 of leukemia cells in vitro.

More preferably, AURKA may be to inhibit KDM6B-mediated p21 expression in leukemia cells in vitro, but is not limited thereto.

In addition, the present invention can provide a method for inhibiting the expression of a protein selected from the group consisting of KDM6B and p21 of leukemia cells comprising the step of treating AURKA with leukemia cells in vitro.

The expression suppression method may be to suppress the differentiation of leukemia cells in vitro, but is not limited thereto.

“KDM6B” used in the present invention may be (NCBI NO.NM_001080424).

“AURKA” used in the present invention may be (NCBI accession no.NM_198433.3).

Hereinafter, examples will be described in detail to help understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

<Experimental Example>

The following experimental examples are intended to provide an experimental example commonly applied to each embodiment according to the present invention.

1. Cell culture

THP-1 cells were grown in RPMI-1640 medium, HEK 293T cells were in Dulbecco's modified Eagle's medium containing 10% inactivated fetal bovine serum and 0.05% penicillin-streptomycin at 37 ° C. in 5% CO ₂ atmosphere. Grown.

For differentiation, THP-1 cells (2 × 10 ⁷ ) were seeded in 100 mm plates and treated with 100 ng / ml PMA (Sigma-Aldrich) or DMSO (Duksan). Cells were collected for the experiment after 48 hours of incubation.

For inhibition of AURKA or KDM6B, THP-1 cells (4 × 10 ⁶ ) were seeded in 60 mm plates and treated with 0.3 μM alisertib (LKT Laboratories) or 5 μM GSK-J4 (Cayman Chemical).

After incubation for 24 or 48 hours, cells were collected and used in the experiment.

2. Plasmid Construction

Plasmids pCMV3-Flag-GATA1 and -YY1 (Han et al., 2015; Son et al., 2012), pGFP-AURKA (Kim et al., 2016a) and pGL3-p21 were prepared by previously reported methods. The KDM6B promoter region was amplified by the KDM6B promoter region from human genomic DNA using the primer pair shown in Table 1, and then inserted into the Nhe I / Hin dIII position of the pGL3-Basic vector (Promega).

The shRNAs for human AURKA and KDM6B were designed using siRNA sequence designer software (Clontech). The double template oligonucleotide for constructing the shRNA plasmid was prepared as shown in Table 1 using a 5'-to-3 'primer.

The oligonucleotide was inserted into the Age I / Eco RI position of the pLKO.1 TRC vector.

3. Western Blot

Total protein was isolated from cells using RIPA solution (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% SDS, 0.5% SDC, 1% NP-40, and 1 mM EDTA), and each antibody Western blot analysis was performed using.

4. Antibodies

GAPDH (CSB-PA00025A0Rb; Cus Ab); AURKA (sc-373856), YY1 (sc-7341), p21 (sc-397), and H3 (sc-8654) (Santa Cruz Biotechnology); H3K4me2 (07-030), H3K9me2 (07-441), H3K27me2 (07-452) and H3K36me2 (07-274) (Millipore); And H3S10ph (9701S) (Cell Signaling Technology) for Western blot and ChIP analysis.

5. Histone tablets

After the cells were collected and washed once with PBS, the cell pellet was resuspended in 1 ml of TEB lysis solution (0.5% Triton ^TM X-100, 2 mM phenylmethylsulfonyl fluoride and 1 × protease inhibitor cocktail), and incubated at 4 ° C. for 30 minutes. It promoted hypotonic swelling.

After recovering the cells by centrifugation, they were re-suspended with 400 μl of 0.4 N H2SO4, and incubated overnight at 4 ° C in a stirrer.

After centrifugation, the supernatant was transferred and 132 μl of 100% TCA was added and treated at 4 ° C. for 2 hours. The pellet was obtained by centrifugation again, washed twice with acetone, and the pellet was resuspended with distilled water.

6. Quantitative real-time PCR

Total RNA was isolated from cells using RNAiso Plus (TaKaRa). For the synthesis of complementary DNA (cDNA), 1 μg of RNA and oligo dT primers (Invitrogen) were incubated at 70 ° C for 5 minutes to denature.

According to the manufacturer's instructions, cDNA was synthesized with MMLV reverse transcriptase. In order to inactivate the reverse transcriptase catalytic activity, cDNA was incubated at 95 ° C for 30 seconds. Synthesized cDNA was used for mRNA expression analysis.

Quantitative real time PCR (Quantitative real time-PCR; qRT-PCR) was performed using SYBR® Green Supermix kit (TaKaRa), and amplification reaction was performed under the following conditions: denaturation at 94 ° C, Table 1 Annealing at a specific temperature for each primer such as (annealing), and the extension (extension) process at 72 ℃ 39 cycles.

The average threshold cycle (Ct) and standard error value were calculated from each Ct value obtained from three reactions per step.

The average Ct value normalized to ΔCt subtracted from the average Ct of GAPDH was estimated, and the ΔΔCt value was calculated as the difference between the control ΔCt and the value obtained in each sample.

The n-fold change in gene expression compared to the control was calculated as 2 ^-ΔΔCt .

7. Chromatin immunoprecipitation analysis (ChIP)

THP-1 cells (1 × 10 ⁷ ) were dispensed into 100 mm plates and treated with 0.3 μM alisertib or DMSO. After 24 hours, 1% formaldehyde was treated with the medium for 10 minutes at room temperature to crosslink the cells with 1% formaldehyde. Then 125 mM glycine was treated at room temperature for 5 minutes and dissolved by adding SDS dissolution solution (1% SDS, 10 mM EDTA, and 50 mM Tris [pH 8.1]).

Samples were sonicated and immunoprecipitated with each antibody. After eluting the immunoprecipitate and cross-linking, the DNA fragment was purified using a PCR purification kit (Axygen).

The precipitated DNA fragment was amplified using a specific primer for the KDM6B promoter region through qRT-PCR, and the primer pairs were as follows: forward, 5'-CTCCCTTTGGGGAAAGCTAA-3 'and reverse, 5'-TGATAAGAGTGCCCGCTACC- 3

The average Ct and standard error values were calculated with each Ct value obtained by two experiments per step, and the standardized average Ct was estimated as ΔCt minus the input average Ct.

8. Flow cytometric analysis of cell differentiation

To confirm cell differentiation, THP-1 cells (1 × 10 ⁶ ) were dispensed into 35 mm dishes and treated with DMSO or 100 ng / ml PMA (phorbol 12-myristate 13-acetate) for 48 hours. Cells were trypsinized, washed and resuspended for 1 hour in cold PBS containing 1 mM EDTA, 1% bovine serum albumin (BSA) and 10 mM sodium azide.

Before cell flow analysis, cells were stained with E-CD11b (12-0118-42) and APC-CD14 (17-0149-42) antibodies for 30 minutes and contained 1 mM EDTA and 1% bovine serum albumin (BSA). After washing with PBS, flow cytometry analysis was performed using a BD Accuri ^TM C6 cytometer (BD Biosciences).

9. Luciferase analysis

For transcriptional activity analysis, HEK 293T cells (2 × 10 ⁴ ) were seeded in 48 well plates, and 24 hours using polyethylenimine (Polysciences), 0.1 or 0.3 μM alisertib or 2 or 5 μM GSK-J4 During the pGL3p21 promoter or pGL3KDM6B promoter reporter plasmid, the appropriate DNA constructs were transfected together.

After transfection, cells were collected to perform luciferase analysis. The level of β-galactosidase activity was used to standardize reporter luciferase. All results were repeated at least 3 times and are shown as representative values.

<Example 1> AURK-mediated H3S10 phosphorylation level reduction during leukemia cell differentiation confirmed

Despite much information on the development of acute myeloid leukemia (AML) rearranged into mixed leukemia (MLL), little research has been conducted on leukemia cell differentiation due to histone modification.

Accordingly, to identify epigenetic changes during the differentiation of the MLL-AF9 AML cell line THP-1 after treatment with PMA (phorbol 12-myristate 13-acetate), the surface markers of macrophages such as CD14 and CD11b (ITGAM) As a result of performing qRT-PCR, the differentiation of THP-1 cells was confirmed as shown in FIG. 1A.

In a previous study, differentiation and maturation of myeloid leukemia has been reported to induce heterochromatin density, and similarly, levels of the closed chromatin markers H3K27me2, H3K27me3 and H3K9me2 are significant during THP-1 differentiation, as shown in Figure 1B. It was confirmed to increase. In addition, interestingly, it was confirmed that the level of H3S10 phosphorylation significantly decreased during the THP-1 differentiation.

According to previous studies, the AURK protein group mainly regulates H3S10 phosphorylation during the cell cycle and regulates gene expression during the HL-60 differentiation period (Crosio et al., 2002; Kim et al., 2016; Ota et al ., 2002), the expression level of AURK family members was confirmed.

As a result, it was confirmed that the mRNA levels of AURKA and AURKB were lower in THP-1 cells treated with PMA than in control cells as shown in FIG. 1C. In addition, down-regulation of AURKA protein by PMA treatment was confirmed in THP-1 cells as shown in FIG. 1D.

According to the above results, in order to confirm what role AURKA plays in THP-1 differentiation, the effects of two different target shRNAs specific for AURKA on the differentiation of THP-1 cells were confirmed.

As a result, similar to the case where PMA was treated as shown in FIG. 1E, knockdown of AURKA induced an increase in CD14 and CD11b levels. In addition, referring to FIG. 1F, it was confirmed that the level of CD14 and CD11b and total histone modification appeared similar to the previous results on the THP-1 cells treated with allertip.

From the above results, it was confirmed that PMA treatment reduced AURKA expression and H3S10 phosphorylation in THP-1 cells, and AURKA could be involved in differentiation.

<Example 2> Confirmation of KDM6B transcription and p21 expression regulation of AURKA

The induction of THP-1 differentiation by alisertib treatment was confirmed, and the effect of AURKA on transcription regulation of the target gene was analyzed.

Using the previously reported microarray results (Shechter et al., 2007), the expression change of leukemia-related genes during THP-1 cell differentiation by PMA was confirmed, and the expression increased according to PMA treatment in THP-1 cells, or After selecting 5 genes showing decreased expression, the expression level of the gene was confirmed by qRT-PCR.

As a result, as shown in FIG. 2A, it was confirmed that the transcription levels of KDM6B, RFX2 and CDK19 were increased in the PMA-treated THP-1 cells, while the levels of CPT1A and SFRP1 were decreased.

In addition, as shown in FIG. 1B, as some histone modifications were identified, including H3S10 phosphorylation and dimethylation of H3K27 and H3K9 in PMA-treated THP-1 cells, concentration of KDM6B expression in THP-1 cell differentiation was concentrated.

First, KDM6B mRNA levels were confirmed in allertip treated THP-1 cells.

As a result, as shown in FIG. 2B, allertip treatment such as PMA treatment increased KDM6B expression level.

Next, to confirm whether AURKA expression decreases the transcriptional activity of KDM6B, a reporter analysis was performed using KDM6B-luc construct on 293T cells.

As a result, as shown in FIG. 2C, allertip induced transcriptional activity in the KDM6B promoter, while AURKA, as shown in FIG. 2D, dose-dependently reduced the KDM6B promoter activity.

According to previous reports, the ectopic expression of p21 was confirmed to undergo monocyte differentiation in peripheral blood monocytes (Asada et al., 1999), confirming that p21 mRNA levels in THP-1 cells can be regulated by AURKA. To do this, qRT-PCR was performed on THP-1 cells treated with allertip.

As a result, as shown in FIG. 2E, the mRNA level of p21 was increased in THP-1 cells treated with allertip.

From the above results, in order to confirm how AURKA regulates p21 transcription level, AURKA transcription activity was confirmed in a dose-dependent manner in the p21 promoter.

As a result, while the promoter activity of p21 was reduced in 293T cells overexpressed with AURKA, as shown in FIG. 2F, the p21 promoter analysis of allertip treated cells promoted promoter p21 transcription according to inactivation of AURKA enzyme function as shown in FIG. 2G. This was confirmed.

From the above results, it was confirmed that AURKA regulates KDM6B and p21 expression during leukemia cell differentiation.

<Example 3> Confirmation of transcriptional inhibition of KDM6B through YY1 recruitment of AURKA

In order to more accurately confirm the KDM6B reduction regulation mechanism by AURKA, transcription factors involved in AURKA-mediated transcription regulation were identified.

As shown in FIG. 3A, the binding sites of GATA1 and YY1 were confirmed in the KDM6B promoter using PROMO to predict transcriptional regulatory factors.

To confirm whether AURKA modulates KDM6B expression in cooperation with GATA1 or YY1, simultaneous immunoprecipitation analysis was performed on 293T cells overexpressing AURKA and YY1 to confirm the correlation between AURKA and YY1.

As a result, it was confirmed that AURKA interacts with YY1 as shown in FIG. 3B. However, the interaction between AURKA and GATA1 was not confirmed in the co-immunoprecipitation analysis as shown in FIG. 3C.

On the other hand, in order to confirm the effect of AURKA and YY1 on KDM6B transcriptional activity, reporter analysis was performed using the KDM6B-luc promoter structure in 293T cells.

As a result, as shown in FIG. 3D, AURKA reduced the transcriptional activity of the KDM6B promoter, and YY1 further reduced the transcriptional activity. In addition, referring to Figure 3E, consistent with the interaction analysis, GATA1 did not show an effect on KDM6B transcriptional activity.

Next, to confirm whether AURKA and YY1 bind to the endogenous promoter regulatory region of the KDM6B gene, ChIP analysis was performed using anti-AURKA and anti-YY1 antibodies after PMA treatment.

As a result, as shown in FIG. 3F, the recruitment of AURKA and YY1 to the target gene promoter was decreased in the cells treated with PMA.

As the alystip shows an effect similar to that of PMA treatment, as a result of performing a ChIP analysis to confirm whether the alys tip reduces the recruitment of YY1 to the KDM6B promoter region, as shown in FIG. 3G, the AURKA inhibitor allistipt is a KDM6B promoter It was confirmed to separate YY1 from the region.

From these results, it can be suggested that KDM6B expression levels are induced through enriched disruption of AURKA and YY1 at the target gene promoter during leukemia cell differentiation.

<Example 4> PMA-dependent p21 activity regulation effect of AURKA through the KDM6B pathway during leukemia cell differentiation

In a previous report, KDM6B H3K27 demethylase activity was confirmed to promote p21 expression in mouse embryonic fibroblasts (Zhao et al., 2013).

Accordingly, to confirm KDM6B-mediated p21 expression, the PMA-treated THP-1 cells were treated with the KDM6B inhibitor GSK-J4 and confirmed p21 expression.

As a result, GSK-J4 significantly reduced p21 levels compared to PMA treatment as shown in FIG. 4A.

To further confirm the transcription control mechanism of p21 through KDM6B, luciferase reporter analysis was performed using the p21-luciferase reporter system in HEK 293T cells.

As a result, as shown in Fig. 4B, ectopic expression of KDM6B promoted p21 transcription, whereas inhibition of KDM6B by GSK-J4, as shown in Fig. 4C, decreased p21 expression.

In addition, it was confirmed that AURKA can reduce p21 transcriptional activity through KDM6B demethylase activity.

As a result, as shown in FIG. 4D, promoter activity of p21 was promoted in THP-1 cells treated with allertip, while the KDM6B inhibitor GSK-J4 inhibited the induction of p21 dependently on the allertip.

To further confirm the change in A21 and KDM6B-dependent p21 expression, the methylation status of the p21 promoter was confirmed through ChIP analysis.

As a result, it was confirmed that the level of H3K27 methylation was decreased in the p21 promoter region of THP-1 cells treated with allertip as shown in FIG. 4E.

From the above results, AURKA function was confirmed as a transcriptional regulator of KDM6B, and it was confirmed that p21 is inactivated after KDM6B inhibition by AURKA.

<Example 5> Confirmation of the effect of KDM6B on the differentiation of THP-1 cells

In order to confirm the role of KDM6B in THP-1 cell differentiation, as a result of treating the KDM6B inhibitor GSK-J4 with THP-1 cells, CD11b and CD14 expression levels were slightly decreased compared to GSK-J4 untreated cells as shown in FIG. 5A. .

To confirm that KDM6B knockdown can inhibit PMA-mediated THP-1 cell differentiation, flow cytometry was performed to confirm expression of monocyte differentiation markers CD11b and CD14.

As a result, it was confirmed that the expression of CD11b and CD14 was increased in THP-1 cells treated with PMA compared to the control cells as shown in FIG. 5B, whereas the CD11b and CD14 of CD11b and CD14 were treated after PMA treatment in THP-1 cells stably expressing KDM6B. Expression showed no change.

In the previous experiments, it was confirmed that allertip induces THP-1 differentiation in mononuclear cells, and thus, flow cytometry was performed to confirm whether KDM6B inhibition can inhibit allertip mediated THP-1 differentiation.

As a result, AURKA inhibition as shown in FIG. 5C increased expression of CD11b and CD14 by inducing THP-1 differentiation. However, GSK-J4 slightly inhibited allertip mediated THP-1 differentiation.

From these results, it was confirmed that KDM6B mediates the differentiation of THP-1 cells through a mechanism dependent on AURKA transcription regulation.

Since the specific parts of the present invention have been described in detail above, for those skilled in the art, it is obvious that this specific technique is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

(1) treating the test substance on leukemia cells;
(2) confirming the binding level of AURKA (Aurora kinase A) and YY1 in leukemia cells treated with the test substance; And
(3) A method for screening for a leukemia therapeutic agent, comprising selecting a test substance that reduces the AURKA and YY1 binding level compared to a control sample. The method of claim 1, wherein the AURKA binds to YY1, and the YY1 bound to AURKA binds to the transcriptional regulatory region of KDM6B. The method according to claim 1, wherein the binding of AURKA and YY1 induces KDM6B expression inhibition, thereby inhibiting KDM6B-mediated p21 expression to inhibit leukemia cell differentiation. The method according to claim 1, wherein the AURKA and YY1 binding level is measured by immunoprecipitation or immunoblotting. It contains AURKA as an active ingredient, and AURKA is a reagent composition for inhibiting the expression of a protein selected from the group consisting of KDM6B and p21 of leukemia cells in vitro. The reagent composition of claim 5, wherein the reagent composition inhibits the differentiation of leukemia cells by inhibiting the expression of a protein selected from the group consisting of KDM6B and p21 of leukemia cells in vitro. A method for inhibiting the expression of a protein selected from the group consisting of KDM6B and p21 of leukemia cells comprising the step of treating AURKA with leukemia cells in vitro. The method according to claim 7, wherein the method for suppressing expression is a method for inhibiting the expression of a protein selected from the group consisting of KDM6B and p21 of leukemia cells, characterized by inhibiting differentiation of leukemia cells in vitro.

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