CG13250, a novel bromodomain inhibitor, suppresses proliferation of multiple myeloma cells in an orthotopic mouse model

Accepted Manuscript CG13250, a novel bromodomain inhibitor, suppresses proliferation of multiple myeloma cells in an orthotopic mouse model Natsuki Imayoshi, Makoto Yoshioka, Jay Chauhan, Susumu Nakata, Yuki Toda, Steve Fletcher, Jeffery W. Strovel, Kazuyuki Takata, Eishi Ashihara PII:

S0006-291X(17)30138-9

DOI:

10.1016/j.bbrc.2017.01.088

Reference:

YBBRC 37150

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 12 January 2017 Accepted Date: 18 January 2017

Please cite this article as: N. Imayoshi, M. Yoshioka, J. Chauhan, S. Nakata, Y. Toda, S. Fletcher, J.W. Strovel, K. Takata, E. Ashihara, CG13250, a novel bromodomain inhibitor, suppresses proliferation of multiple myeloma cells in an orthotopic mouse model, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.01.088. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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CG13250, a novel bromodomain inhibitor, suppresses proliferation of multiple myeloma cells in an orthotopic mouse model

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Natsuki Imayoshi a, Makoto Yoshioka b, Jay Chauhan c, Susumu Nakata d, Yuki Toda a, Steve Fletcher c, Jeffery W. Strovel b, Kazuyuki Takata a, and Eishi Ashihara a, *

Department of Clinical and Translational Physiology, Kyoto Pharmaceutical University,

Kyoto, Japan ConverGene LLC, Gaithersburg, MD, c Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD

Department of Clinical Oncology, Kyoto Pharmaceutical University, Kyoto, Japan

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*Corresponding author Eishi Ashihara, MD, PhD. Address: 5 Nakauchi, Yamashina-ku, Kyoto, 607-8414, Japan e-mail: [email protected] Tel: +81-75-595-4705 Fax: +81-75-595-4796

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Word counts in manuscript text (Abstract, Keywords, Introduction, Materials and Methods, Results, Discussion, Acknowledgements, Conflict of Interest, References, and Figure legends): 4,590 (≤4,600 words) Abstract: 168 (≤250 words) Number of figures/tables: 4 (≤4) Number of supplemental data: one Supplementary material (including 4 figures with the legends and 2 tables)

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Abstract Multiple myeloma (MM) is characterized by the clonal proliferation of

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neoplastic plasma cells. Despite a stream of new molecular targets based on better understanding of the disease, MM remains incurable. Epigenomic abnormalities

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contribute to the pathogenesis of MM. bromodomain 4 (BRD4), a member of the

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bromodomain and extraterminal (BET) family, binds to acetylated histones during M/G1 transition in the cell cycle promoting progression to S phase. In this study, we investigated the effects of a novel BET inhibitor CG13250 on MM cells. CG13250 inhibited ligand binding to BRD4 in a dose-dependent manner and with an IC50 value of

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1.1 µM. It inhibited MM proliferation in a dose-dependent manner and arrested cells in G1, resulting in the induction of apoptosis through caspase activation. CG13250

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inhibited the binding of BRD4 to c-MYC promoter regions suppressing the transcription

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of the c-MYC gene. Administered in vivo, CG13250 significantly prolonged survival of an orthotopic MM-bearing mice. In conclusion, CG13250 is a novel bromodomain inhibitor that is a promising molecular targeting agent against MM.

Keywords: multiple myeloma, bromodomain, c-myc, orthotopic mouse model

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Abbreviations

multiple myeloma

BRD

bromodomain

BET

bromodomain and extraterminal domain

EBV

Ebstein-Barr virus

FBS

fetal bovine serum

PC/SM

penicillin-streptomycin

SPF

Specific pathogen-free

r

recombinant

h

human

PI

propidium iodide

qRT-PCR

quantitative reverse transcription-polymerase chain reaction

CDK6

cyclin-dependent kinase 6

PBS

phosphate buffered saline

NP-40

NonidetP-40

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Ab

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MM

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

ChIP

chromatin immunoprecipitation

NMP

N-Methyl-2-pyrrolidone

PG

propylene glycol

PEG

polyethylene glycol

LCL

lymphoblastoid cell

SE

super-enhancer 3

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standard deviation

Tx

treatment

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1. Introduction

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Multiple myeloma (MM) is a malignant neoplasm of plasma cells that is characterized by clonal proliferation of neoplastic plasma cells, production of

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M-proteins, and associated organ damage including bone fractures, renal failure, anemia,

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and hypercalcemia. Due to the availability of new molecular targeting agents such as proteasome inhibitors and immunomodulatory drugs, and the introduction of autologous stem cell transplantation, the outcomes for MM patients have recently improved [1-3]. However, it remains incurable at present [1], and new molecular targeting agents are

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continually being sought and developed in step with better understanding of the pathogenesis underlying MM [1,4-6].

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Epigenetic mechanisms, consisting of DNA methylation, post-translational

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modifications of histone proteins, and the action of non-coding RNA transcripts, regulate gene expression without alterations in DNA sequence. Disruption of these epigenetic processes leads to altered gene function and tumorigenesis [7], and epigenomic abnormalities contribute to the pathogenesis of MM [8]. Post-translational modifications of histone tails, including methylation, acetylation, phosphorylation, ubiquitination, and sumoylation, affect the interaction of DNA with histones and other

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DNA-binding proteins. Bromodomain and extraterminal domain (BET) family proteins bind covalently to acetylated lysine residues of histones and influence gene transcription

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[9] and bromodomain 4 (BRD4) binds to acetylated histones during the M/G1 transition in the cell cycle promoting progression to S phase [10,11]. Dysregulation of the BET

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family protein functions, for example by oncogenic fusion proteins, leads to the

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development of several types of cancers. Therefore, BET family proteins are recently recognized as a target molecule for cancer therapy.

JQ-1 [12] and I-BET762 [13] were the first BET inhibitors to be described and these compounds suppress MM cell proliferation [14,15]. Other BET inhibitors

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described to date include PFI-1, RVX-208, and OTX015 [9,16]. By conducting chemical screening against BRD4 and subsequent chemical optimization, we developed

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a novel bromodomain inhibitor CG13250. Unlike previously described BET inhibitors,

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it has a quinolinone core and displayed high affinity and specificity to BET proteins (S. Fletcher, et al. In preparation). In this study, we investigated the inhibitory effects of CG13250 on the proliferation of MM cells.

2. Materials and Methods

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2.1. Cell lines, cell culture, reagents, and animals Human AMO-1, NCI-H929, and OPM-2 MM cell lines, and the IM-9

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Ebstein-Barr virus (EBV) -transformed cell line derived from a patient with MM, were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH

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(Braunschweig, Germany). MM.1s cell line was purchased from American Type Culture

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Collection (Manassas, VA). NCI-H929, OPM-2, MM.1s, and IM-9 cells were cultured in RPMI1640 (Wako Pure Chemical Industries, Osaka, Japan) containing 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO) and 1% penicillin-streptomycin (PC/SM; Wako Pure Chemical Industries). The AMO-1 cell line

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was cultured in RPMI1640 containing 20% FBS and 1% PC/SM. For in vivo experiments, we established IM-9Luc cells. About 2 × 106 IM-9 cells were transfected

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with 2 µg of a pGL4.51[luc/CMV/Neo] vector (Promega, Tokyo, Japan) using a

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Ncleofector 2b device (Lonza, Basel, Switzerland). Transfected cells were cultured in regular media overnight and then were cultured in RPMI1640 containing 10% FBS, 1% PC/SM, and 600 µg/mL of geneticin (Wako Pure Chemical Industries). All cell lines were maintained at 37°C in a fully humidified atmosphere of 20% O2, 5% CO2, and 75% N2. BET inhibitor JQ-1 was purchased from Cayman Chemical (Ann Arbor, MI). Specific pathogen-free (SPF) 8–10-week-old female SCID mice (Clea Japan, Tokyo,

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Japan) were used for the in vivo experiments. Approval for these studies was obtained

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from the Committee on Animal Research of Kyoto Pharmaceutical University.

2.2. AlphaScreen assay

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To identify compounds that inhibit the binding of BET ligands to the

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bromodomain, we utilized alphaScreen technology (PerkinElmer, Waltham, MA) and BRD4 bromodomain ligand (BPS Bioscinece, San Diego, CA). All of the binding reactions were conducted at room temperature. For the inhibition of BRD4-binding reactions, the reaction mixture comprised the supplied assay buffer containing 2.5 nM

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recombinant human (rh) BRD4-BD1-BD2 (BPS Bioscience) and the indicated amount of CG13250. The reaction mixture was incubated for 30 min followed by additional 30

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min incubation after the addition of rh BRD4-BD1-BD2. Then, BRD detection buffer

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containing 10 µg/mL glutathione acceptor beads and 10 µg/mL streptavidin donor beads was added and the final mixture was incubated for 50 min in a dark room. AlphaScreen signal was measured using an EnSpire Alpha 2390 Multilabel reader (PerkinElmer). The percent activity in the presence of CG13250 was calculated according to the following equation: % activity = [(A- Ab)/(At - Ab)]×100, where A = the AlphaScreen signal in the presence of the compound, Ab = the AlphaScreen signal in the absence of the BET

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Ligand, and At = the AlphaScreen signal in the absence of the compound. The percent inhibition was calculated according to the following equation: % inhibition = 100 - %

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activity.

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2.3. Growth inhibitory effects on MM cells

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Cell proliferation was evaluated by a WST-8 assay using Cell Count Reagent SF (Nakalai Tesque, Kyoto, Japan), as previously described [17]. MM cells were seeded in a flat-bottomed 96-well plate (BD Bioscience, Tokyo, Japan) at a density of 2 × 104 cells in 100 µL of medium per well, and were then incubated with serial dilutions of

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evaluated.

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JQ-1 or CG13250 for 72 h. The mean of four samples at each concentration was

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2.4. Cell cycle analysis and induction of apoptosis Cell cycle analysis using propidium iodide (PI) and flow cytometry was performed as previously described [17]. Apoptosis induced by CG13250 was determined using an Annexin-V-FITC Apoptosis Detection Kit I (BD Bioscience), according to the manufacturer’s instructions. Cells were analyzed with a FACS Calibur using CellQuest software (BD Bioscience). Data were analyzed with FlowJo software (FlowJo LLC,

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Ashland, OR).

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2.5. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

RNA was purified with the illustra RNAspin Mini RNA Isolation Kit (GE

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Healthcare Japan, Tokyo, Japan) and subjected to reverse transcription. Human c-MYC,

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CCND1, p21, and 18s ribosomal RNA mRNA expression levels were measured by real-time PCR. Each real-time PCR reaction mixture contained 20 µL of Taqman master mix (Roche Diagnostics GmbH Mannheim, Germany), cDNA, pairs of primers, and Taqman probe (Universal Probe Library, Roche Diagnostics GmbH). The cDNA was

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amplified with a Thermal Cycler Dice system (Takara Bio, Kusatsu, Japan) using the following parameters: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and

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60°C for 60 s. To normalize for loading difference, 18s ribosomal RNA mRNA was used

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as an internal control. The primers used in this study are shown in Table S1.

2.6. Western blot analysis Following treatment with JQ-1 and CG13250 compound, expression levels of c-MYC, cyclin D1, cyclin-dependent kinase 6 (CKD6), and caspase-3 were examined by Western blotting. More than 1 × 106 cells were collected by centrifugation, and then

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the cells were washed with ice-cold phosphate buffered saline (PBS) twice. Preparation of cell lysates, immunoblotting, and detection of bands corresponding to

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immunoreactive proteins were performed as previously described [18]. Protein concentrations were determined using a Quantus Fluorometer (Promega, Tokyo, Japan).

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Samples were analyzed using the following primary antibodies (Abs), as indicated:

anti-caspase-3,

anti-cleaved

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anti-c-MYC (Santa Cruz Biotechnology, Dallas, TX), anti-cyclin D1 (BD Bioscience), caspase-3,

anti-CKD6,

and

anti-glyceraldehyde

3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology Japan, Tokyo, Japan).

as secondary Ab.

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Horseradish peroxidase-coupled IgG (Amersham Biosciences, Tokyo, Japan) was used

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2.7. Chromatin immunoprecipitation assay

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IM-9 cells were treated with JQ-1 or CG13250 for 24 h. After the treatment, the chromatin in the cells was cross-linked with 1% formaldehyde at room temperature for 5 min. The cells were then washed three times for 5 min in ice-cold PBS. BRD4 chromatin immunoprecipitation (ChIP) analysis was carried out as follows: Cross-linked cells were lysed with cell lysis buffer (5 mM HEPES pH 8.0, 85 mM KCl, 0.5% NonidetP-40 (NP-40), and proteinase inhibitor (Sigma-Aldrich)) and subsequently,

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the nuclear extracts were lysed with nucleus lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS, and proteinase inhibitor). The nuclear extracts were sonicated

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with a Bioruptor (Cosmo Bio, Tokyo, Japan) and were resuspended in dilution buffer (0.01% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, and proteinase

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inhibitor). The sonicated cell lysates were incubated overnight at 4°C with 5 µg of anti-BRD4 Ab (Bethyl Labs, Montgomery, MA) and then, the lysates were incubated

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for 2 h with 50 µL of Dynabeads (Thermo Fisher Scientific) in dilution buffer. Beads were washed once with wash buffer 1 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0, and 150 mM NaCl), once with wash buffer 2 (0.1% SDS, 1%

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Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0, and 500 mM NaCl), once with LiCl wash buffer (10 mM Tris pH 8.0, 1 mM EDTA, 250 mM LiCl, 1% NP-40, 1%

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Na-deoxycholate), and twice with Tris-EDTA buffer. DNA was eluted in elution buffer

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(50 mM Tris pH 8.0, 10 mM EDTA, and 1% SDS) overnight at 65°C. The eluted samples were treated with RNase A and Proteinase K to digest RNA and protein, and then DNA was purified by phenol chloroform extraction and ethanol precipitation. Binding of BRD4 to the c-MYC promoter and c-MYC enhancer in the chromatin immunoprecipitates was analyzed by SYBR Green real-time PCR using a Thermal Cycler Dice (Takara Bio). Fold enrichment of c-MYC promoter and c-MYC enhancer

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DNA in the chromatin immunoprecipitates was determined from triplicate PCR reactions at two c-MYC promoter and c-MYC enhancer regions over the amount of input

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DNA normalized against the amount of c-MYC promoter and c-MYC enhancer DNA in

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the input samples [19]. The oligos used for this analysis are shown in Table S2.

2.8. In vivo effects of CG13250 in a MM-bearing mouse model

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The in vivo inhibitory effects of CG13250 on IM-9Luc cells were investigated using an orthotopic MM mouse model as previously described [17]. After exposure to 2 Gy of irradiation, SPF SCID mice (Japan Clea, Osaka, Japan) were inoculated with 2 ×

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106 IM-9Luc cells in 100 µL of PBS through the tail vein. CG13250 was dissolved in N-Methyl-2-pyrrolidone (NMP) just before administration. CG13250 solubilized in

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NMP was diluted with propylene glycol (PG), polyethylene glycol (PEG) 400, and H2O

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in a volume ratio of 10/40/50 of NMP/PG/H2O. Mice were orally administered with vehicle or CG13250 (50 mg/kg/dose) in a cycle of 3 doses in 2 days (morning; night; morning) for 20 cycles, starting the day after inoculation of IM-9Luc cells. MM growth was investigated using the in vivo Lumina IIIXR imaging system (PerkinElmer) twice a week.

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2.8. Statistical analysis

value of 0.05 was considered statistically significant.

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3. Results

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The in vivo effects of CG13250 treatment were analyzed using the log-rank test. A p

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3.1. CG13250 inhibits the proliferation of MM cells by induction of apoptosis By conducting chemical screening against BRD4 and subsequent chemical optimization, we developed a novel bromodomain inhibitor CG13250 with a quinolinone core. CG13250 inhibited the binding of histone H4 peptide ligand to

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tandem bromodomains of BRD4 in a dose-dependent manner, and with an IC50 of 1.1 µM (Fig. 1A). We next investigated the inhibitory effects of CG13250 on MM cells

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using a WST-8 assay. We used JQ-1 as a preexisting BRD4 inhibitor. Similar to JQ-1

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(Fig. S1), CG13250 inhibited growth of five MM cell lines in a dose-dependent manner (Fig. 1B). We analyzed the effect of JQ-1 and CG13250 on the cell cycle in two of the cell lines, NCI-H929 and OPM-2, by flow cytometry. CG13250 arrested the cell cycle of MM cells, and the proportion in G1 phase was increased in a time-dependent manner (Fig. 1C, 1D, and Fig. S2). We then examined the induction of apoptosis in CG13250-treated cells by Annexin-V/PI staining. The proportion of early apoptotic

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cells (Annexin-V+/PI-), late apoptotic cells, and necrotic cells (Annexin-V+/PI+)

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increased in a dose-dependent manner (Fig. 1D, 1E, and Fig. S3).

3.2. Alteration of c-MYC and cell cycle-regulating proteins by CG13250 treatment

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We next investigated alterations in c-MYC and cell cycle-regulating protein

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expression in MM cells. Analysis by qRT-PCR in NCI-H929 and OPM-2 cells demonstrated that CG13250 treatment caused a decrease in the level of c-MYC mRNA transcripts (Fig. 2A). CCND1 mRNA was not expressed in OPM-2 cells [20,21], but that in NCI-H929 cells was also decreased (Fig. 2A). By contrast, p21 mRNA

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transcripts were increased by CG13250 treatment (Fig. 2A). In Western blot analyses, the levels of c-MYC were decreased by CG13250 treatment (Fig. 2B). To a varying

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degree, cyclin D1, and CKD6 proteins were also decreased (cyclin D1 proteins did not

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express in OPM-2 cells, Fig. 2B). The levels of cleaved caspase-3 protein were clearly increased by CG13250 treatment (Fig. 2B). Taken together, these results indicate that, accompanied by a cell cycle arrest in G1, CG13250 suppressed c-MYC transcription in MM cells, and consequently induced apoptosis through the activation of caspase-3.

3.3. CG13250 inhibits the binding of BRD4 to c-MYC promoters and c-MYC enhancers

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Analysis of c-MYC mRNA transcript levels in IM-9 cells by qRT-PCR demonstrated that they were decreased by CG13250 treatment in a dose-dependent

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manner (Fig. 3A), and Western blot analysis showed that c-MYC protein levels had decreased by 48 h after CG13250 treatment (Fig. 3B). To investigate whether CG13250

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inhibited the binding of BRD4 to c-MYC promoters, we performed a ChIP assay using

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IM-9 cells after immunoprecipitation with an anti-BRD4 Ab. CG13250 decreased BRD4 binding to c-MYC promoter and enhancer regions in a dose-dependent manner (Fig. 3C).

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3.4. CG13250 prolongs survival of MM-bearing mice Lastly, we assessed the in vivo effects of CG13250 using an IM-9Luc

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cell-bearing mice. After exposure to 2 Gy of irradiation, IM-9Luc cells were inoculated

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through the tail vein. All animals died by approximately 30 days after transplantation. IM-9Luc cells had engrafted in many organs including in the bone marrow of the spine and femurs, and in the kidneys, ovaries, and stomach (Fig. S4). CG13250 treatment resulted in reduced luminescence intensity of IM-9Luc cells on day 15 after inoculation (Fig. 4A) and prolonged the survival of mice (mean, 33.7 days) compared to the vehicle-control arm (mean, 27.1 days; p=0.029; Fig. 4B). There were no statistical

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differences in body weight during the treatment (data not shown).

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4. Discussion

Since the discovery of JQ-1 and I-BET762, many compounds that inhibit the binding of

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BET proteins to acetylated histones have been developed. Most, including JQ-1,

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I-BET762, OTX015, and CPI-203 have a triazolodiazepine-based structure. PFI-1 and RVX-208 have a quinazolinone-based structure [9,16]. By conducting chemical screening against BRD4 and subsequent chemical optimization, we developed a novel bromodomain inhibitor CG13250. Unlike previously described bromodomain inhibitors,

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it has a quinolinone core and displayed high affinity and specificity to BET proteins (S. Fletcher, et al. In preparation). We demonstrated that CG13250 suppresses MM cell

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proliferation both in vitro and in vivo.

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CG13250 inhibited the binding of BRD4 to its ligand in a dose-dependent manner. The BET family consists of the four ubiquitously expressed BRD2, BRD3 and BRD4, plus BRDT that is specifically expressed in testis. Each BET protein has two highly conserved bromodomains: BD1 and BD2, which bind to acetylated lysines on histones. A BROMOscan assay [22] shows that the dissociation constant for CG13250 and BRD4 is 79 nM (S. Fletcher, et al. In preparation). This value is similar to those of

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previously developed BRD4 inhibitors [9,16]. However, with a quinolinone core, the chemical structure of CG13250 is different from those earlier compounds.

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We investigated the in vitro inhibitory effects of CG13250 against four MM cell lines and IM-9 cell line. IM-9 cell line is EBV-transformed B-lymphoblastoid cell

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(LCL) line derived from a patient with MM, which has been historically used as a MM

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cell line [17,23-25]. CG13250 inhibited the proliferation of MM cells in a dose-dependent manner. Notwithstanding any differences in their sensitivity, proliferation of all cell lines tested was inhibited by CG13250 treatment. To study the mechanism by which CG13250 reduced the cell proliferation, we examined the effects

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on cell cycle progression and the expression of cell cycle regulator molecules. Flow cytometric analysis showed that CG13250 induced a cell cycle arrest in G1 phase in a

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time-dependent manner. Western blot and qRT-PCR analyses revealed that expression of

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driver molecules of the G1/S transition cyclin D1 and CDK6 were decreased and the CDK inhibitor p21 was increased by the CG13250 treatment. Furthermore, CG13250 induced both early (Annexin-V+/PI- fraction) and late (Annexin-V+/PI+ fraction) phases of apoptosis. As Western blotting also showed the cleavage of caspase-3 in CG13250-treated MM cells and Giemsa staining revealed fragmentation of condensed nuclei (data not shown), we conclude that CG13250 induces apoptosis through the

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activation of caspases. c-MYC, a master regulator of cell proliferation, is dysregulated in the advanced

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stages of MM [26], and proliferation of MM cells results from addiction to c-MYC proteins [27], making c-MYC a promising therapeutic intervention point in MM. In the

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present study, we found that CG13250 suppressed c-MYC mRNA transcripts as well as

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c-MYC proteins in MM cells. Moreover, a ChIP assay demonstrated that the binding of BRD4 was reduced at c-MYC promoter and enhancer regions in IM-9 cells by the treatment of CG13250. It has been recently demonstrated that BRD4 inhibition reduced the proliferation of LCLs [28,29]. Moreover, EBV super-enhancers (ESEs) are essential

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for EBV-transformed LCL growth and BRD4 protein binds to ESEs and activates MYC expression in LCLs, which is a similar observation that BRD4 binds to the

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super-enhancer regions in MM cells [30]. Taken together, these findings indicate that

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CG13250 suppresses BRD4 binding to c-MYC promoter and enhancer regions and inhibits c-MYC expression in MM cells. CG13250 decreases cyclin D1 expression leading to cell cycle arrest in G1, resulting in the induction of apoptosis by the activation of caspase-3. Lastly, we investigated the in vivo inhibitory effects of CG13250 using an IM-9Luc cells. IM-9 cells have been used historically for establishment of a MM mouse

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model [17,25]. In vivo imaging of orthotopic IM-9Luc-bearing MM mice demonstrated that CG13250 treatment reduced the luminescence intensity in the early phase (day 15)

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of the treatment duration and prolonged survival compared to controls. Moreover, major complications such as body weight loss or diarrhea did not develop. Taken together,

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these observations suggest that the oral administration of a novel bromodomain

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inhibitor, CG13250, may represent an effective strategy against MM.

In conclusion, CG13250 is a bromodomain inhibitor with a novel structure that has promise as a molecular targeting agent against MM. In future communications, we

Conflict of Interest

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plan to report biological characterizations of the derivatives of CG13250.

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N. Imayoshi, S. Nakata, J. Chauhan, Y. Toda, S. Flether, K. Takata, and E. Ashihara

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have no financial conflict of interest to disclose. M. Yoshioka and J.W. Strovel are employees of ConverGene LLC.

Acknowledgements This work was partly supported by Grant-in-Aids for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT,

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26461436 to EA, 16K08722 to SN, and 16K08286 to KT) and MEXT-Supported Program for the Strategic Research Foundation at Private Universities, 2017-2021

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(S1511024L to EA).

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References

review about the future, J. Hematol. Oncol. 9 (2016) 52.

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[1] L. Naymagon, M. Abdul-Hay, Novel agents in the treatment of multiple myeloma: a

SC

[2] B. Bjorkstrand, G. Gahrton, High-dose treatment with autologous stem cell

(2007) 227-233.

M AN U

transplantation in multiple myeloma: past, present, and future, Semin. Hematol. 44

[3] P.G. Richardson, C. Mitsiades, R. Schlossman, et al., New drugs for myeloma, Oncologist 12 (2007) 664-689.

TE D

[4] E. Ashihara, T. Takada, T. Maekawa, Targeting the canonical Wnt/beta-catenin pathway in hematological malignancies, Cancer Sci. 106 (2015) 665-671.

EP

[5] N.A. Keane, S.V. Glavey, J. Krawczyk, et al., AKT as a therapeutic target in multiple

AC C

myeloma, Expert Opin. Ther. Targets 18 (2014) 897-915. [6] E.M. Ocio, P.G. Richardson, S.V. Rajkumar, et al., New drugs and novel mechanisms of action in multiple myeloma in 2013: a report from the International Myeloma Working Group (IMWG), Leukemia 28 (2014) 525-542. [7] A.P. Feinberg, B. Tycko, The history of cancer epigenetics, Nat. Rev. Cancer 4 (2004) 143-153.

22

ACCEPTED MANUSCRIPT

[8] K. Dimopoulos, P. Gimsing, K. Gronbaek, The role of epigenetics in the biology of multiple myeloma, Blood Cancer J. 4 (2014) e207.

acetylation, Nat. Rev. Drug Discov. 13 (2014) 337-356.

RI PT

[9] P. Filippakopoulos, S. Knapp, Targeting bromodomains: epigenetic readers of lysine

SC

[10] K. Mochizuki, A. Nishiyama, M.K. Jang, et al., The bromodomain protein Brd4

Chem. 283 (2008) 9040-9048.

M AN U

stimulates G1 gene transcription and promotes progression to S phase, J. Biol.

[11] X. Gao, X. Wu, X. Zhang, et al., Inhibition of BRD4 suppresses tumor growth and

(2016) 679-685.

TE D

enhances iodine uptake in thyroid cancer, Biochem. Biophys. Res. Commun. 469

[12] P. Filippakopoulos, J. Qi, S. Picaud, et al., Selective inhibition of BET

EP

bromodomains, Nature 468 (2010) 1067-1073.

AC C

[13] E. Nicodeme, K.L. Jeffrey, U. Schaefer, et al., Suppression of inflammation by a synthetic histone mimic, Nature 468 (2010) 1119-1123.

[14] J.E. Delmore, G.C. Issa, M.E. Lemieux, et al., BET bromodomain inhibition as a therapeutic strategy to target c-Myc, Cell 146 (2011) 904-917. [15] A. Chaidos, V. Caputo, K. Gouvedenou, et al., Potent antimyeloma activity of the novel bromodomain inhibitors I-BET151 and I-BET762, Blood 123 (2014)

23

ACCEPTED MANUSCRIPT

697-705. [16] M. Brand, A.M. Measures, B.G. Wilson, et al., Small molecule inhibitors of

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bromodomain-acetyl-lysine interactions, ACS Chem. Biol. 10 (2015) 22-39.

[17] H. Yao, E. Ashihara, J.W. Strovel, et al., AV-65, a novel Wnt/beta-catenin signal

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inhibitor, successfully suppresses progression of multiple myeloma in a mouse

M AN U

model, Blood. Cancer J. 1 (2011) e43.

[18] H. Fukuda, S. Nakamura, Y. Chisaki, et al., Daphnetin inhibits invasion and migration of LM8 murine osteosarcoma cells by decreasing RhoA and Cdc42 expression, Biochem. Biophys. Res. Commun. 471 (2016) 63-67.

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[19] W. Fiskus, Y. Wang, A. Sreekumar, et al., Combined epigenetic therapy with the histone methyltransferase EZH2 inhibitor 3-deazaneplanocin A and the histone

EP

deacetylase inhibitor panobinostat against human AML cells, Blood 114 (2009)

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2733-2743.

[20] J. Glassford, N. Rabin, E.W. Lam, et al., Functional regulation of D-type cyclins by insulin-like growth factor-I and serum in multiple myeloma cells, Br. J. Haematol. 139 (2007) 243-254. [21] K. Specht, E. Haralambieva, K. Bink, et al., Different mechanisms of cyclin D1 overexpression in multiple myeloma revealed by fluorescence in situ hybridization

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and quantitative analysis of mRNA levels, Blood 104 (2004) 1120-1126. [22] M.A. Fabian, W.H. Biggs, 3rd, D.K. Treiber, et al., A small molecule-kinase

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interaction map for clinical kinase inhibitors, Nat. Biotechnol. 23 (2005) 329-336. [23] G.W. Rhyasen, M.M. Hattersley, Y. Yao, et al., AZD5153: A Novel Bivalent BET

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Bromodomain Inhibitor Highly Active against Hematologic Malignancies, Mol.

M AN U

Cancer Ther. 15 (2016) 2563-2574.

[24] J. Qian, J. Xie, S. Hong, et al., Dickkopf-1 (DKK1) is a widely expressed and potent tumor-associated antigen in multiple myeloma, Blood 110 (2007) 1587-1594.

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[25] P. Beauparlant, D. Bedard, C. Bernier, et al., Preclinical development of the nicotinamide phosphoribosyl transferase inhibitor prodrug GMX1777, Anticancer

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Drugs 20 (2009) 346-354.

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[26] W.M. Kuehl, P.L. Bergsagel, Multiple myeloma: evolving genetic events and host interactions, Nat. Rev. Cancer 2 (2002) 175-187.

[27] T. Holien, T.K. Vatsveen, H. Hella, et al., Addiction to c-MYC in multiple myeloma, Blood 120 (2012) 2450-2453. [28] J. Liang, H. Zhou, C. Gerdt, et al., Epstein-Barr virus super-enhancer eRNAs are essential for MYC oncogene expression and lymphoblast proliferation, Proc. Natl.

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Acad. Sci. U. S. A. 113 (2016) 14121-14126. [29] H. Zhou, S.C. Schmidt, S. Jiang, et al., Epstein-Barr virus oncoprotein

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super-enhancers control B cell growth, Cell Host Microbe 17 (2015) 205-216.

[30] J. Loven, H.A. Hoke, C.Y. Lin, et al., Selective inhibition of tumor oncogenes by

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disruption of super-enhancers, Cell 153 (2013) 320-334.

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Figure Legends

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Fig. 1. The effects of CG13250 on MM cell proliferation. (A) AlphaScreen assay for CG13250. CG13250 inhibited the binding of histone H4 peptide ligand to tandem

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bromodomains of BRD4 in a dose-dependent manner. Results shown represent the

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means ± standard deviation (SD) of two independent experiments. (B) WST-8 assay for detecting the effects of CG13250 on the MM cell proliferation. Data represent the mean ± standard error of three independent experiments each with four replicates experiments. (C), (D) Cell cycle states of NCI-H929 (C) and OPM-2 (D) cells by CG13250 or JQ-1

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treatment were assessed. CG13250 arrested the cell cycle of MM cells with the increase of the proportion in G1 phase. Data represent the mean ± SD of three independent

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experiments. Solid, open, gray, and dotted bars indicate sub G1, G0/G1, S, and G2/M

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phases of the cell cycle, respectively. (E), (F) Induction of apoptosis in NCI-H929 (E) and OPM-2 (F) cells were assessed. CG13250 induced apoptosis in MM cells. Data represent the mean + SD of three independent experiments. Open, gray, and solid bars indicate the percentages of vehicle-, JQ-1-, and CG13250-treated cells, respectively. No Tx, no treatment.

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Fig. 2. Alteration of c-MYC and cell cycle-related molecules by CG13250 treatment. (A) MM cells were incubated with CG13250 or JQ-1 at the indicated concentrations and

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times, and then qRT-PCR was performed. Results shown are representative of three independent experiments. (B) Cells were incubated with CG13250 or JQ-1 at the

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indicated concentrations and times, and then western blotting was performed. Results

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shown are representative of two or three independent experiments. No Tx, no treatment.

Fig. 3. Alteration of c-MYC expression in IM-9 cells by CG13250 treatment. (A), (B) IM-9 cells were incubated with CG13250 or JQ-1 at the indicated concentrations and

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times, and then qRT-PCR to detect c-MYC mRNA transcripts (A) and western blotting to detect c-MYC proteins (B) were performed. Results shown are representative of three

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independent experiments. (C) A ChIP assay using an anti-BRD4 antibody. Fold

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enrichment of real-time PCR values of the precipitated c-MYC promoter and enhancer regions bound to the BRD4 proteins normalized by the values of no treatment control are shown. No Tx, no treatment.

Fig. 4. The in vivo effects of CG13250 administration. The experimental procedures are described in the Materials and Methods section. (A) Bioluminescent images of IM-9Luc

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cells on 15 days after inoculation. (B) The in vivo inhibitory effects of CG13250 on IM-9Luc cells using an orthotopic mouse model. Survival of IM-9Luc-bearing mice

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treated with CG13250 was significantly prolonged compared to vehicle-treated mice (p=0.029). Solid and dashed lines represent the survival rates of the CG13250-treated

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group (ten mice) and the vehicle-treated group (seven mice), respectively.

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Highlights

A novel bromodomain inhibitor CG13250 suppresses MM cell proliferation.




CG13250 decreases C-MYC expression, resulting in the induction of apoptosis.




CG13250 prolongs the survivals of MM-bearing mice.

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