- Email: [email protected]
HDAC inhibitor against diffuse large B-cell lymphoma
Accepted Manuscript BEBT-908: A novel potent PI3K/HDAC inhibitor against diffuse large B-cell lymphoma Jin Li, Changgeng Qian, Qianqian Zhou, Jiwei Li, Kunlun Li, Pingyong Yi PII:
S0006-291X(17)31498-5
DOI:
10.1016/j.bbrc.2017.07.139
Reference:
YBBRC 38245
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 19 July 2017 Revised Date:
0006-291X June 0006-291X
Accepted Date: 25 July 2017
Please cite this article as: J. Li, C. Qian, Q. Zhou, J. Li, K. Li, P. Yi, BEBT-908: A novel potent PI3K/ HDAC inhibitor against diffuse large B-cell lymphoma, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.07.139. 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.
ACCEPTED MANUSCRIPT BEBT-908: A Novel Potent PI3K/HDAC Inhibitor against Diffuse Large B-cell Lymphoma
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Jin Lia, Changgeng Qianb, Qianqian Zhoua, Jiwei Lia, Kunlun Lia, Pingyong Yia,*
Department of Hematology, The Affiliated Cancer Hospital of Xiangya School of Medicine, Central
South University, Changsha, Hunan, 410006, China.
Guangzhou BeBetter Medicine Technology Co., Ltd, Guangzhou, Guangdong, 510000, China.
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b
*
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Corresponding author: Dr. Pingyong Yi, Department of Hematology, The Affiliated Cancer Hospital of
Xiangya School of Medicine, Central South University, 283 Tongzipo Road, Changsha, Hunan, 410006,
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China. E-mail: [email protected]. Tel: +86 137 8616 1716.
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ACCEPTED MANUSCRIPT BEBT-908: A Novel Potent PI3K/HDAC Inhibitor against Diffuse Large B-cell Lymphoma
Abstract
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The PI3K pathway and epigenetic regulation have been shown to play a pivotal role in the development and progression of diffuse large B-cell lymphoma (DLBCL). In the clinic, existing PI3K and HDAC inhibitors show limited efficacy as single agents toward DLBCL. However, in preclinical studies, the
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synergistic effects of PI3K inhibitors and HDAC inhibitors on DLBCL have sparked the enthusiasm of
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researchers to target both PI3K and HDAC. We hypothesized that a novel dual PI3K/HDAC inhibitor, BEBT-908, would display improved pharmacologic effects on DLBCL. We analyzed the anti-DLBCL activity of BEBT-908 in a comprehensive manner, demonstrating its role in the suppression of in vitro cell proliferation, blockade of PI3K and HDAC activities, inhibition of multiple signaling pathways, and
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promotion of apoptosis and cell cycle arrest. BEBT-908 showed potent PI3K/HDAC inhibition, with nanomolar IC50 values against DLBCL cell lines. Moreover, BEBT-908 inhibited multiple pathways, including JAK/STAT3, MAPK/ERK and NF-κB, and induced apoptosis and cell cycle arrest at G1 phase in
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these cells. Additionally, dual PI3K/HDAC inhibition was superior to the inhibition of PI3K or HDAC
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alone. The dual inhibitor BEBT-908 is a promising lead compound for developing novel targeted therapeutic agents against DLBCL.
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Keywords: PI3K inhibitor, HDAC inhibitor, diffuse large B-cell lymphoma
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Abbreviations: DLBCL, diffuse large B-cell lymphoma; NHL, non-Hodgkin lymphoma; GCB, germinal center B-cell-like; ABC, activated B-cell-like; PI3K, phosphatidylinositol-3-kinase; BCL, B-cell lymphoma; CLL/SLL, chronic lymphocytic leukemia/small lymphocytic lymphoma; ORR, objective
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remission rate; HAT, histone acetyltransferase; HDAC, histone deacetylases; TCL, T-cell lymphoma;
Chemical compounds studied in this article Vorinostat (PubChem CID: 5311)
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Idelalisib (PubChem CID: 11625818)
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SAHA, suberoylanilide hydroxamic acid; PBMC: peripheral blood mononuclear cells.
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1. Introduction Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma (NHL),
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accounting for nearly 40% of all new cases of lymphoma every year[1]. DLBCL is a heterogeneous malignancy in terms of its genetic, pathological, and clinical features[2]. It can be categorized into germinal center B-cell-like (GCB) and activated B-cell-like (ABC) DLBCL on the basis of gene
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expression profiles[3]. Although the standard regimen R-CHOP has greatly improved the prognosis of
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DLBCL patients, one-third of patients with advanced diseases eventually become refractory or experience relapse[4]. Thus, there is an urgent need for developing better therapeutic methods based on the molecular pathogenesis of DLBCL.
The phosphatidylinositol-3-kinase (PI3K) pathway plays a major role in several physiological functions,
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including protein synthesis, proliferation, differentiation, motility, autophagy and apoptosis[5]. Activation of the PI3K signaling pathway is a feature of numerous human malignancies, including indolent B-cell lymphoma (BCL) and aggressive BCL[6]. The loss of the tumor suppressor gene PTEN is the primary
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mechanism of PI3K pathway activation in GCB DLBCL, whereas the constitutive activation of B-cell
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receptor (BCR) signaling plays a dominant role in other DLBCL subtypes[6,7]. The PI3K family is divided into three classes, namely, class I, II, and III. Class I PI3Ks are composed of regulatory (p85) and catalytic (p110) subunits. The p110 subunit comprises four isoforms (α, β, δ and γ)[8]. The α and β isoforms are ubiquitously expressed in all tissues, while the δ and γ isoforms are mainly expressed in hematologic cells[9]. Idelalisib, a PI3Kδ inhibitor, was approved in 2014 by the US Food and Drug Administration (FDA) for the treatment of relapsed chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Despite resulting in a promising objective remission rate (ORR) of 47%-57% in patients with 4
ACCEPTED MANUSCRIPT indolent BCL (CLL/SLL, follicular lymphoma and mantle cell lymphoma), idelalisib did not show clinical activity in DLBCL in a phase I study[10,11,12]. Dysregulation of epigenetics, including DNA modification and protein modification, as well as
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non-coding RNA regulation, are common in many human cancers. Histone acetylation is one of the epigenetic modifications that alters DNA transcription and protein expression by modifying the structural relationship between histone proteins and DNA[13]. A balance between histone acetyltransferases (HATs)
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and histone deacetylases (HDACs) regulates histone acetylation. The increase in histone acetylation by
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HATs promotes DNA transcription by loosening chromatins, whereas the decrease in histone acetylation by HDACs leads to the inhibition of gene expression via chromatin compaction[14]. Overexpression and aberrant activity of HDACs in hematological malignancies lead to the dysregulation of DNA transcription and gene expression[15]. This has provided the theoretical basis for developing HDAC inhibitors as a
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novel antitumor therapeutic approach. HDAC inhibitors suppress the activity of HDACs to transform proteins from a deacetylated to an acetylated state. This results in the differentiation, growth arrest, immunogenicity and apoptosis of tumor cells and inhibits angiogenesis[16]. Three HDAC inhibitors,
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namely, the pan-HDAC inhibitor vorinostat, the class I selective HDAC inhibitor romidepsin and the
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pan-HDAC inhibitor belinostat, are currently approved by the US FDA for the treatment of relapsed or refractory T-cell lymphoma (TCL). Another class I/II-specific HDAC inhibitor panobinostat is approved for the treatment of relapsed or refractory multiple myeloma[17]. However, HDAC inhibitors have limited efficacy as single agents against DLBCL and solid tumors[18,19]. Currently, PI3K inhibitors and HDAC inhibitors are often largely ineffective as monotherapies for the treatment of DLBCL. Evidence in the literature provides a rationale to target both PI3Ks and HDACs in solid tumors and hematopoietic malignancies[20,21]. BEBT-908 is a novel dual PI3K/HDAC inhibitor that 5
ACCEPTED MANUSCRIPT is currently being tested in a phase I clinical trial in China (CTR20160223). We analyzed the anti-DLBCL activity of BEBT-908 in a comprehensive manner, demonstrating its role in the suppression of in vitro cell proliferation, blockade of PI3K and HDAC activities, inhibition of multiple signaling pathways, and
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cytotoxic effect on and induced apoptosis in DLBCL cell lines.
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promotion of apoptosis and cell cycle arrest. Our results showed that treatment with BEBT-908 exerted a
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2. Materials and Methods
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2.1 Chemicals and reagents BEBT-908 was kindly provided by BeBetter Medicine Technology Co., Ltd. (Guangzhou, China). Suberoylanilide hydroxamic acid (SAHA or vorinostat) and CAL-101 (idelalisib) were purchased from
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Selleck, China. For in vitro studies, all compounds were dissolved in DMSO and stored at -80°C. Primary
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antibodies against p-AKT (Ser473), AKT, p-STAT3 (Tyr705), STAT3, p-Erk1/2 (Thr202/Tyr204), ERK, NF-κB p65, IκBα, Bcl-2, cleaved-PARP, p21 and GAPDH were obtained from Cell Signaling Technologies, China. Primary antibodies against Histone H3 (acetyl K9 + K14 + K18 + K23 + K27),
2.2 Cell lines and culture
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Histone H3 and Lamin B were obtained from Abcam, China.
The human ABC DLBCL cell line SU-DHL-2 and human GCB DLBCL cell lines (SU-DHL-6 and
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Pfeiffer) were obtained from American Type Culture Collection (ATCC). TMD8 (human ABC DLBCL
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cells) and OCI-LY-7 (human GCB DLBCL cells) were gifts from BeBetter Medicine Technology Co., Ltd. Peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood of healthy volunteers using Ficoll-Paque centrifugation. OCI-LY-7 cells were cultured in IMDM (Gibco) supplemented with 20% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and the other cell lines were cultured in RPMI-1640 (Gibco) medium containing 10% FBS and 1% penicillin-streptomycin. All cells were grown at 37°C in a humidified incubator with 5% CO2.
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ACCEPTED MANUSCRIPT 2.3 Cell growth inhibition assay Cells were seeded in opaque walled 96-well plates (3×104 cells/well) and treated with SAHA, CAL-101 or BEBT-908 at various concentrations for 24 hours, 48 hours and 72 hours. Growth inhibition was
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assayed based on cellular ATP content using the CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega).
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2.4 Western blot analysis
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For total protein extraction, we used RIPA buffer (Beyotime) supplemented with protease inhibitor phenylmethylsulfonyl fluoride (PMSF; Beyotime) and phosphatase inhibitor (Roche) to lyse cells following treatment. Histone proteins and nuclear and cytoplasmic proteins were extracted using the EpiQuik Total Histone Extraction Kit (EpiGentek) and Nuclear and Cytoplasmic Extraction Kit
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(Beyotime), respectively, following the manufacturer’s instructions. The BCA protein assay kit (Beyotime) was used to measure protein concentration. Protein samples were separated via SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The blots were blocked with 5% non-fat
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milk and incubated with specific primary antibodies overnight at 4°C. Protein bands were detected with the
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ECL Western Blotting Substrate (Millipore).
2.5 Flow cytometric analysis for the detection of apoptosis and cell cycle arrest Cells were seeded in 6-well plates, incubated with SAHA, CAL-101 or BEBT-908 for 24 hours and then harvested for the detection of apoptosis or cell cycle distribution. Apoptosis and cell cycle distribution were analyzed using the Annexin V-FITC Apoptosis Detection Kit (BD Biosciences) according to the manufacturer’s instructions. All data were acquired using BD FACSCalibur. All data were analyzed using 8
ACCEPTED MANUSCRIPT CellQuest Pro and ModFit LT.
2.6 Statistical analysis
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SPSS 24.0 was used to analyze the data. All data are presented as the means ± SD. Statistics were
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performed using one-way ANOVA. P-values of <0.05 were considered statistically significant.
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3.1 BEBT-908 effectively inhibits the growth of DLBCL cells
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3. Results
CellTiter-Glo® assay was performed to compare the effect of SAHA, CAL-101 and BEBT-908 on
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cellular proliferation in a panel of 5 DLBCL cell lines. All three drugs decreased the viability of lymphoma
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cell in a time- and dose-dependent manner. BEBT-908 displayed potent antiproliferative activity (average IC50 = 0.025 µM, 0.013 µM and 0.009 µM for 24 hours, 48 hours and 72 hours, respectively) in all tested DLBCL cell lines. Notably, BEBT-908 was more potent than SAHA (average IC50 = 1.471 µM, 0.830 µM and 0.754 µM for 24 hours, 48 hours and 72 hours, respectively) and CAL-101 (average IC50 = 22.815
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µM, 12.224 µM and 6.339 µM for 24 hours, 48 hours and 72 hours, respectively) (Table 1). At the 72-hour timepoint, the average IC50 values for the individual lymphoma sub-groups were 0.010 µM (0.008–0.011 µM) for ABC DLBCL cell line and 0.009 µM (0.003–0.019 µM) for GCB DLBCL cell lines (Fig. 1B).
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cells.
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BEBT-908 exhibited no significant difference in growth inhibition between the two sub-groups of DLBCL
To ensure that the antiproliferative activity of BEBT-908 was not because of general cytotoxicity, we tested its growth inhibition effect on PBMCs. The results showed that PBMCs were considerably resistant to the antiproliferative effect of BEBT-908 (IC50 = 23.274 µM) (Table 1). Thus, BEBT-908 exerts potent antiproliferative effect specifically on DLBCL cells.
Table 1 Growth inhibition of DLBCL cell lines by BEBT-908 and reference compounds 10
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CAL-101 (µM)
BEBT-908 (µM)
Cell line 48h
72h
24h
48h
72h
24h
48h
72h
PBMC
-
-
315.13
-
-
890.908
-
-
23.274
TMD8
1.801
0.966
0.742
12.531
2.277
1.198
0.024
0.011
0.008
SU-DHL-2
1.702
0.784
0.809
43.559
27.593
9.852
SU-DHL-6
2.091
1.385
1.258
34.285
14.317
7.959
OCI-LY-7
1.386
0.774
0.674
22.434
16.737
12.607
Pfeiffer
0.373
0.239
0.289
1.266
0.196
0.014
0.011
0.047
0.027
0.019
0.017
0.008
0.006
0.009
0.004
0.003
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0.027
0.079
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PBMC: peripheral blood mononuclear cell
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24h
3.2 BEBT-908 is a potent inhibitor of both PI3K and HDAC and abrogates key DLBCL signaling pathways
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via HDAC inhibition
We conducted Western blot analysis to detect the expression level of p-AKT in the five DLBCL cell lines. Among the cell lines, the highest level of p-AKT was observed in SU-DHL-2 and Pfeiffer cells
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corresponding to ABC- and GCB- DLBCL cells, respectively (Fig. 2A). Based on the results of growth
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inhibitory effects of BEBT-908 and p-AKT expression level in the cell lines, SU-DHL-2 and Pfeiffer cells were selected for subsequent experiments. To confirm that BEBT-908 deactivates PI3K and HDAC in vitro, we further examined its effects in SU-DHL-2 and Pfeiffer cell lines. BEBT-908 inhibited p-AKT expression in a dose-dependent manner, and its inhibitory effect was clearly superior to that of CAL-101 (Fig. 2B and C). An increase in the level of Ac-H3 was also detected in a dose-dependent manner in response to BEBT-908 treatment. Notably, BEBT-908 was more potent than SAHA at deactivating HDAC (Fig. 2D and E). Therefore, BEBT-908 is a 11
ACCEPTED MANUSCRIPT potent inhibitor of both PI3K and HDAC. To investigate whether BEBT-908 suppresses other growth and survival pathways via feedback mechanisms, SU-DHL-2 and Pfeiffer cells were treated with SAHA, CAL-101 or BEBT-908. In ABC-
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(e.g., SU-DHL-2) and GCB- DLBCL cells (e.g., Pfeiffer), the JAK/STAT3 and MAPK/ERK pathway were activated, which resulted in resistance of these cells to CAL-101. In contrast, BEBT-908 and SAHA inhibited the p-STAT3 and p-ERK1/2 in these lymphoma cells (Fig. 2F and G). Therefore, HDAC
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inhibition by BEBT-908 effectively downregulates the JAK/STAT3 and MAPK/ERK pathways.
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The NF-κB pathway is a key pathway in ABC DLBCL cells. We found that BEBT-908 decreased the levels of nuclear p65 but increased the levels of cytoplasmic p65 and IκBα in SU-DHL-2 (Fig. 2F), thereby inhibiting the NF-κB signaling pathway. Therefore, BEBT-908 abrogates multiple signaling pathways
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likely via HDAC inhibition.
3.3 BEBT-908 induces apoptosis in DLBCL cells
BEBT-908 was shown to induce cell death, promote cell cycle arrest or both. Therefore, we used
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Annexin- V/PI staining to investigate the effects of BEBT-908 on apoptosis in DLBCL cells. BEBT-908
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increased the proportion of apoptotic cells, including early and late apoptotic cells in a dose-dependent manner. In addition, it was more potent than SAHA and CAL-101 at the same dose (Fig. 3A and B). While there were approximately 5.00% and 5.68% apoptotic cells in untreated SU-DHL-2 and Pfeiffer cell lines, respectively, the percentage of apoptotic cells increased to 40.34% and 46.45% in these respective cell lines in response to 100 nM BEBT-908 (Fig. 3B). To elucidate the possible mechanism by which BEBT-908 induced apoptosis, we performed Western blot analysis of apoptotic markers. The increase in apoptosis induction in SU-DHL-2 and Pfeiffer cells following treatment with increasing doses of 12
ACCEPTED MANUSCRIPT BEBT-908 was confirmed via analysis of PARP cleavage and Bcl-2 reduction. Moreover, BEBT-908 increased PARP cleavage and decreased Bcl-2 levels to a greater extent than the same concentration of SAHA and CAL-101 did (Fig. 3C). Thus, BEBT-908 likely induces apoptosis in DLBCL cells by
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activating mitochondrial-mediated pathways.
3.4 BEBT-908 arrests the cell cycle at G1 phase in DLBCL cells
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Next, we studied the effect of BEBT-908 on cell cycle progression of DLBCL cells using PI staining.
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Consistent with earlier reports with other HDAC inhibitors[22], we observed that BEBT-908 and SAHA induced cell cycle arrest at G1 phase. In contrast, the same concentration of CAL-101 had no significant effect on the cell cycle. In addition, the proportion of cells arrested at the G1 phase increased with the dose of BEBT-908 (Fig. 4A and B). In control groups of SU-DHL-2 and Pfeiffer cells, only 22.94% and 29.16%
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cells were in G1 phase, respectively, whereas in 100 nM BEBT-908-treated groups of SU-DHL-2 and Pfeiffer cells, the proportion of cells at the G1 phase significantly increased to 66.21% and 66.54%, respectively. Furthermore, while 58.46% and 53.63% of cells were in S phase in the control groups of
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SU-DHL-2 and Pfeiffer cells, respectively, and treatment with 100 nM BEBT-908 reduced the proportion
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to 22.17% and 20.96%, respectively (Fig. 4B). The increase in the proportion of cells in the G1 phase and the decrease in the proportion of cells in S phase in response to BEBT-908 were consistent with the dose-dependent induction of p21 expression. In addition, BEBT-908 induced p21 expression more potently than did SAHA and CAL-101, which was consistent with the flow cytometry results. (Fig. 4C). Therefore, BEBT-908 exerts its inhibitory effects in different DLBCL cell lines (Table 1 and Fig. 1B, CellTiter-Glo® assay) by both inducing apoptosis and promoting cell cycle arrest.
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Discussion
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At present, a growing number of PI3K inhibitors and HDAC inhibitors are being widely tested in
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clinical trials or have been approved for use in the clinic. These compounds show promising activities in indolent BCL and TCL, although they have limited efficacy on DLBCL. A phase II clinical trial of a combination of idelalisib and entospletinib for the treatment of relapsed and refractory NHL demonstrated only 17% ORR in DLBCL[23]. A phase II trial of vorinostat in relapsed DLBCL resulted in only 5.6%
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ORR[24]. Another phase II clinical trial with a pan-HDAC inhibitor panobinostat in relapsed DLBCL displayed 29% ORR[25]. Despite these outcomes, the combination of PI3K/mTOR inhibitor BEZ235 and the HDAC inhibitor panobinostat was found to synergistically promote cell death in DLBCL irrespective
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of histological types[26]. In addition, the simultaneous inhibition of the PI3K/mTOR signaling pathway
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and HDAC with rapamycin and panobinostat in DLBCL cells resulted in growth inhibition and apoptosis of tumor cells[27]. Based on this evidence, we demonstrated that BEBT-908, a novel dual PI3K/HDAC inhibitor, exerts a potent activity against diverse DLBCL cell lines in vitro. In this study, we found that the administration of very low (nanomolar) concentrations of BEBT-908 suppressed cell proliferation, inhibited AKT phosphorylation and increased histone H3 acetylation in ABCand GCB- DLBCL cell lines but not in normal PBMCs, highlighting the potential therapeutic selectivity of BEBT-908. Notably, the level of AKT phosphorylation was independent of the sensitivity of DLBCL cell 14
ACCEPTED MANUSCRIPT lines to BEBT-908. Thus, AKT phosphorylation may not be the only determining factor of the responsiveness of DLBCL cells to BEBT-908, although a high basal p-AKT level might indicate an increase in the dependence of cells on the PI3K/AKT pathway for survival. BEBT-908 induced apoptosis,
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PARP cleavage, Bcl-2 downregulation, G1 cell cycle arrest and p21 accumulation in DLBCL cells. HDAC inhibitors typically induce G1 cell cycle arrest through the upregulation of the cyclin-dependent kinase inhibitor p21[28]. HDAC inhibitors can also activate the mitochondrial-mediated apoptotic pathway and
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regulate the equilibrium between pro- and anti-apoptotic proteins[22,29]. BEBT-908 was more potent than
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SAHA and CAL-101. The low efficacy of SAHA and CAL-101 might explain the poor outcomes of DLBCL patients in response to these agents in clinical trials.
We also found that BEBT-908 inhibited multiple signaling pathways associated with DLBCL oncogenesis. Apart from suppressing the activation of its direct targets, namely PI3K and HDAC,
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BEBT-908 also effectively reduced the activation of JAK/STAT3, MAPK/ERK, and NF-κB, none of which are the direct targets of BEBT-908. Similar effects were observed in response to SAHA. HDAC inhibitors lead to the acetylation of histone and non-histone protein, which subsequently regulate the activity or
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expression of functional proteins, including STAT3, NF-κB, HIF-1α, c-Myc, p53[30,31].
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Therefore, the synchronous effect on multiple targets, such as PI3K and HDAC, may be a more efficient approach than acting on single targets. The major advantages of a single multi-targeted inhibitor over the combination of several single targeted drugs are simplified pharmacokinetics, easy administration, reduced side effects and improved patient compliance. In addition, it is more effective to combine a single multi-targeted inhibitor with other novel small molecule inhibitors or chemotherapeutic drugs for further clinical development. In conclusion, we have discovered a highly selective and potent dual PI3K/HDAC inhibitor, BEBT-908, 15
ACCEPTED MANUSCRIPT which displays better antiproliferative effects against DLBCL cells than single targeted PI3K and HDAC inhibitors. BEBT-908 thus represents a novel and alternative approach to enhance the activity of PI3K and HDAC inhibitors, and it warrants further clinical investigation to be validated as a therapeutic agent for
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DLBCL.
Acknowledgements
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None.
Funding
This work was supported by the Guangzhou BeBetter Medicine Technology Co., Ltd.
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Conflict of interest
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There are no potential conflicts of interest to disclose
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[21] X. Liu, A. Wang, X. Liang, J. Liu, F. Zou, C. Chen, Z. Zhao, Y. Deng, H. Wu, Z. Qi, B. Wang, L. Wang, F. Liu, Y. Xu, W. Wang, S.M. Fernandes, R.M. Stone, I.A. Galinsky, J.R. Brown, T. Loh, J.D. Griffin, S. Zhang, E.L. Weisberg, X. Zhang, J. Liu, Q. Liu, Simultaneous inhibition of Vps34 19
ACCEPTED MANUSCRIPT kinase would enhance PI3Kdelta inhibitor cytotoxicity in the B-cell malignancies, Oncotarget 7 (2016) 53515-53525. [22] H. Rikiishi, Autophagic and apoptotic effects of HDAC inhibitors on cancer cells, J Biomed
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Biotechnol 2011 (2011) 830260. [23] P.M. Barr, G.B. Saylors, S.E. Spurgeon, B.D. Cheson, D.R. Greenwald, S.M. O'Brien, A.K. Liem, R.E. McLntyre, A. Joshi, E. Abella-Dominicis, M.J. Hawkins, A. Reddy, J. Di Paolo, H. Lee, J. He, J.
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rituximab in relapsed diffuse large B-cell lymphoma, Blood 128 (2016) 185-194. [26] M. Rahmani, M.M. Aust, E.C. Benson, L. Wallace, J. Friedberg, S. Grant, PI3K/mTOR inhibition markedly potentiates HDAC inhibitor activity in NHL cells through BIM- and MCL-1-dependent mechanisms in vitro and in vivo, Clin Cancer Res 20 (2014) 4849-4860.
[27] M. Gupta, S.M. Ansell, A.J. Novak, S. Kumar, S.H. Kaufmann, T.E. Witzig, Inhibition of histone deacetylase overcomes rapamycin-mediated resistance in diffuse large B-cell lymphoma by inhibiting Akt signaling through mTORC2, Blood 114 (2009) 2926-2935. 20
ACCEPTED MANUSCRIPT [28] V.Y. Yazbeck, S. Grant, Romidepsin for the treatment of non-Hodgkin's lymphoma, Expert Opin Investig Drugs 24 (2015) 965-979. [29] Y. Zhao, J. Tan, L. Zhuang, X. Jiang, E.T. Liu, Q. Yu, Inhibitors of histone deacetylases target the
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[31] L. Ellis, H. Hammers, R. Pili, Targeting tumor angiogenesis with histone deacetylase inhibitors,
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Figure 1. Growth inhibition of DLBCL cell lines by BEBT-908. A, Structure of BEBT-908. B, IC50 curves
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in 5 DLBCL cell lines after 24 hours, 48 hours and 72 hours of BEBT-908 exposure.
Figure 2. Inhibition of PI3K, HDAC and multiple signaling pathways by BEBT-908. SU-DHL-2 and Pfeiffer cells were treated with DMSO, 100 nM SAHA, 100 nM CAL-101, and 100 nM BEBT-908 or with
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DMSO and increasing concentrations of BEBT-908 for 24 hours. A, Expression of p-AKT in 5 DLBCL cell lines. B and C, Inhibition of p-AKT. D and E, Increase of Ac-H3. F, Inhibition of JAK/STAT3,
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in Pfeiffer.
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MAPK/ERK and NF-κB pathways in SU-DHL-2. G, Inhibition of JAK/STAT3 and MAPK/ERK pathways
Figure 3. BEBT-908 induces apoptosis. SU-DHL-2 and Pfeiffer cells were treated with DMSO, 100 nM SAHA, 100 nM CAL-101, and 100 nM BEBT-908 or with DMSO and increasing concentrations of BEBT-908 for 24 hours. A, Representative histograms of Annexin-V /PI staining assessed using flow cytometry. B, Percentage of apoptotic cells (including early and late apoptotic cells) determined using flow cytometry. *p<0.05 vs control. C, Western blot analysis for apoptotic markers in cell lysates.
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ACCEPTED MANUSCRIPT Figure 4. BEBT-908 induces G1 phase cell cycle arrest. SU-DHL-2 and Pfeiffer cells were treated with DMSO, 100 nM SAHA, 100 nM CAL-101, and 100 nM BEBT-908 or with DMSO and increasing concentrations of BEBT-908 for 24 hours. Flow cytometry analysis using PI staining to assess cell cycle
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distribution. A, Representative histograms of cell cycle distribution assessed by PI staining. B, Bar graphs depicting the relative percentage of cells in each phase of the cell cycle in response to BEBT-908 and
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reference compounds. *p<0.05 vs control. C, Western blot analysis of p21 expression.
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ACCEPTED MANUSCRIPT Highlights 1. BEBT-908 exerts cytotoxic and antiproliferative effects on DLBCL. 2. BEBT-908 represses the PI3K/AKT pathway and HDAC activity in DLBCL cells.
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4. BEBT-908 induces apoptosis and G1 phase arrest in DLBCL cells.
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3. BEBT-908 inhibits key DLBCL pathways, including JAK/STAT3, MAPK/ERK and NF-κB.
S0006-291X(17)31498-5
DOI:
10.1016/j.bbrc.2017.07.139
Reference:
YBBRC 38245
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 19 July 2017 Revised Date:
0006-291X June 0006-291X
Accepted Date: 25 July 2017
Please cite this article as: J. Li, C. Qian, Q. Zhou, J. Li, K. Li, P. Yi, BEBT-908: A novel potent PI3K/ HDAC inhibitor against diffuse large B-cell lymphoma, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.07.139. 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.
ACCEPTED MANUSCRIPT BEBT-908: A Novel Potent PI3K/HDAC Inhibitor against Diffuse Large B-cell Lymphoma
a
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Jin Lia, Changgeng Qianb, Qianqian Zhoua, Jiwei Lia, Kunlun Lia, Pingyong Yia,*
Department of Hematology, The Affiliated Cancer Hospital of Xiangya School of Medicine, Central
South University, Changsha, Hunan, 410006, China.
Guangzhou BeBetter Medicine Technology Co., Ltd, Guangzhou, Guangdong, 510000, China.
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b
*
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Corresponding author: Dr. Pingyong Yi, Department of Hematology, The Affiliated Cancer Hospital of
Xiangya School of Medicine, Central South University, 283 Tongzipo Road, Changsha, Hunan, 410006,
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China. E-mail: [email protected]. Tel: +86 137 8616 1716.
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ACCEPTED MANUSCRIPT BEBT-908: A Novel Potent PI3K/HDAC Inhibitor against Diffuse Large B-cell Lymphoma
Abstract
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The PI3K pathway and epigenetic regulation have been shown to play a pivotal role in the development and progression of diffuse large B-cell lymphoma (DLBCL). In the clinic, existing PI3K and HDAC inhibitors show limited efficacy as single agents toward DLBCL. However, in preclinical studies, the
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synergistic effects of PI3K inhibitors and HDAC inhibitors on DLBCL have sparked the enthusiasm of
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researchers to target both PI3K and HDAC. We hypothesized that a novel dual PI3K/HDAC inhibitor, BEBT-908, would display improved pharmacologic effects on DLBCL. We analyzed the anti-DLBCL activity of BEBT-908 in a comprehensive manner, demonstrating its role in the suppression of in vitro cell proliferation, blockade of PI3K and HDAC activities, inhibition of multiple signaling pathways, and
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promotion of apoptosis and cell cycle arrest. BEBT-908 showed potent PI3K/HDAC inhibition, with nanomolar IC50 values against DLBCL cell lines. Moreover, BEBT-908 inhibited multiple pathways, including JAK/STAT3, MAPK/ERK and NF-κB, and induced apoptosis and cell cycle arrest at G1 phase in
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these cells. Additionally, dual PI3K/HDAC inhibition was superior to the inhibition of PI3K or HDAC
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alone. The dual inhibitor BEBT-908 is a promising lead compound for developing novel targeted therapeutic agents against DLBCL.
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Keywords: PI3K inhibitor, HDAC inhibitor, diffuse large B-cell lymphoma
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Abbreviations: DLBCL, diffuse large B-cell lymphoma; NHL, non-Hodgkin lymphoma; GCB, germinal center B-cell-like; ABC, activated B-cell-like; PI3K, phosphatidylinositol-3-kinase; BCL, B-cell lymphoma; CLL/SLL, chronic lymphocytic leukemia/small lymphocytic lymphoma; ORR, objective
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remission rate; HAT, histone acetyltransferase; HDAC, histone deacetylases; TCL, T-cell lymphoma;
Chemical compounds studied in this article Vorinostat (PubChem CID: 5311)
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Idelalisib (PubChem CID: 11625818)
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SAHA, suberoylanilide hydroxamic acid; PBMC: peripheral blood mononuclear cells.
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1. Introduction Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma (NHL),
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accounting for nearly 40% of all new cases of lymphoma every year[1]. DLBCL is a heterogeneous malignancy in terms of its genetic, pathological, and clinical features[2]. It can be categorized into germinal center B-cell-like (GCB) and activated B-cell-like (ABC) DLBCL on the basis of gene
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expression profiles[3]. Although the standard regimen R-CHOP has greatly improved the prognosis of
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DLBCL patients, one-third of patients with advanced diseases eventually become refractory or experience relapse[4]. Thus, there is an urgent need for developing better therapeutic methods based on the molecular pathogenesis of DLBCL.
The phosphatidylinositol-3-kinase (PI3K) pathway plays a major role in several physiological functions,
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including protein synthesis, proliferation, differentiation, motility, autophagy and apoptosis[5]. Activation of the PI3K signaling pathway is a feature of numerous human malignancies, including indolent B-cell lymphoma (BCL) and aggressive BCL[6]. The loss of the tumor suppressor gene PTEN is the primary
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mechanism of PI3K pathway activation in GCB DLBCL, whereas the constitutive activation of B-cell
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receptor (BCR) signaling plays a dominant role in other DLBCL subtypes[6,7]. The PI3K family is divided into three classes, namely, class I, II, and III. Class I PI3Ks are composed of regulatory (p85) and catalytic (p110) subunits. The p110 subunit comprises four isoforms (α, β, δ and γ)[8]. The α and β isoforms are ubiquitously expressed in all tissues, while the δ and γ isoforms are mainly expressed in hematologic cells[9]. Idelalisib, a PI3Kδ inhibitor, was approved in 2014 by the US Food and Drug Administration (FDA) for the treatment of relapsed chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Despite resulting in a promising objective remission rate (ORR) of 47%-57% in patients with 4
ACCEPTED MANUSCRIPT indolent BCL (CLL/SLL, follicular lymphoma and mantle cell lymphoma), idelalisib did not show clinical activity in DLBCL in a phase I study[10,11,12]. Dysregulation of epigenetics, including DNA modification and protein modification, as well as
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non-coding RNA regulation, are common in many human cancers. Histone acetylation is one of the epigenetic modifications that alters DNA transcription and protein expression by modifying the structural relationship between histone proteins and DNA[13]. A balance between histone acetyltransferases (HATs)
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and histone deacetylases (HDACs) regulates histone acetylation. The increase in histone acetylation by
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HATs promotes DNA transcription by loosening chromatins, whereas the decrease in histone acetylation by HDACs leads to the inhibition of gene expression via chromatin compaction[14]. Overexpression and aberrant activity of HDACs in hematological malignancies lead to the dysregulation of DNA transcription and gene expression[15]. This has provided the theoretical basis for developing HDAC inhibitors as a
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novel antitumor therapeutic approach. HDAC inhibitors suppress the activity of HDACs to transform proteins from a deacetylated to an acetylated state. This results in the differentiation, growth arrest, immunogenicity and apoptosis of tumor cells and inhibits angiogenesis[16]. Three HDAC inhibitors,
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namely, the pan-HDAC inhibitor vorinostat, the class I selective HDAC inhibitor romidepsin and the
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pan-HDAC inhibitor belinostat, are currently approved by the US FDA for the treatment of relapsed or refractory T-cell lymphoma (TCL). Another class I/II-specific HDAC inhibitor panobinostat is approved for the treatment of relapsed or refractory multiple myeloma[17]. However, HDAC inhibitors have limited efficacy as single agents against DLBCL and solid tumors[18,19]. Currently, PI3K inhibitors and HDAC inhibitors are often largely ineffective as monotherapies for the treatment of DLBCL. Evidence in the literature provides a rationale to target both PI3Ks and HDACs in solid tumors and hematopoietic malignancies[20,21]. BEBT-908 is a novel dual PI3K/HDAC inhibitor that 5
ACCEPTED MANUSCRIPT is currently being tested in a phase I clinical trial in China (CTR20160223). We analyzed the anti-DLBCL activity of BEBT-908 in a comprehensive manner, demonstrating its role in the suppression of in vitro cell proliferation, blockade of PI3K and HDAC activities, inhibition of multiple signaling pathways, and
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cytotoxic effect on and induced apoptosis in DLBCL cell lines.
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promotion of apoptosis and cell cycle arrest. Our results showed that treatment with BEBT-908 exerted a
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2. Materials and Methods
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2.1 Chemicals and reagents BEBT-908 was kindly provided by BeBetter Medicine Technology Co., Ltd. (Guangzhou, China). Suberoylanilide hydroxamic acid (SAHA or vorinostat) and CAL-101 (idelalisib) were purchased from
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Selleck, China. For in vitro studies, all compounds were dissolved in DMSO and stored at -80°C. Primary
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antibodies against p-AKT (Ser473), AKT, p-STAT3 (Tyr705), STAT3, p-Erk1/2 (Thr202/Tyr204), ERK, NF-κB p65, IκBα, Bcl-2, cleaved-PARP, p21 and GAPDH were obtained from Cell Signaling Technologies, China. Primary antibodies against Histone H3 (acetyl K9 + K14 + K18 + K23 + K27),
2.2 Cell lines and culture
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Histone H3 and Lamin B were obtained from Abcam, China.
The human ABC DLBCL cell line SU-DHL-2 and human GCB DLBCL cell lines (SU-DHL-6 and
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Pfeiffer) were obtained from American Type Culture Collection (ATCC). TMD8 (human ABC DLBCL
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cells) and OCI-LY-7 (human GCB DLBCL cells) were gifts from BeBetter Medicine Technology Co., Ltd. Peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood of healthy volunteers using Ficoll-Paque centrifugation. OCI-LY-7 cells were cultured in IMDM (Gibco) supplemented with 20% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and the other cell lines were cultured in RPMI-1640 (Gibco) medium containing 10% FBS and 1% penicillin-streptomycin. All cells were grown at 37°C in a humidified incubator with 5% CO2.
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ACCEPTED MANUSCRIPT 2.3 Cell growth inhibition assay Cells were seeded in opaque walled 96-well plates (3×104 cells/well) and treated with SAHA, CAL-101 or BEBT-908 at various concentrations for 24 hours, 48 hours and 72 hours. Growth inhibition was
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assayed based on cellular ATP content using the CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega).
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2.4 Western blot analysis
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For total protein extraction, we used RIPA buffer (Beyotime) supplemented with protease inhibitor phenylmethylsulfonyl fluoride (PMSF; Beyotime) and phosphatase inhibitor (Roche) to lyse cells following treatment. Histone proteins and nuclear and cytoplasmic proteins were extracted using the EpiQuik Total Histone Extraction Kit (EpiGentek) and Nuclear and Cytoplasmic Extraction Kit
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(Beyotime), respectively, following the manufacturer’s instructions. The BCA protein assay kit (Beyotime) was used to measure protein concentration. Protein samples were separated via SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The blots were blocked with 5% non-fat
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milk and incubated with specific primary antibodies overnight at 4°C. Protein bands were detected with the
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ECL Western Blotting Substrate (Millipore).
2.5 Flow cytometric analysis for the detection of apoptosis and cell cycle arrest Cells were seeded in 6-well plates, incubated with SAHA, CAL-101 or BEBT-908 for 24 hours and then harvested for the detection of apoptosis or cell cycle distribution. Apoptosis and cell cycle distribution were analyzed using the Annexin V-FITC Apoptosis Detection Kit (BD Biosciences) according to the manufacturer’s instructions. All data were acquired using BD FACSCalibur. All data were analyzed using 8
ACCEPTED MANUSCRIPT CellQuest Pro and ModFit LT.
2.6 Statistical analysis
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SPSS 24.0 was used to analyze the data. All data are presented as the means ± SD. Statistics were
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performed using one-way ANOVA. P-values of <0.05 were considered statistically significant.
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3.1 BEBT-908 effectively inhibits the growth of DLBCL cells
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3. Results
CellTiter-Glo® assay was performed to compare the effect of SAHA, CAL-101 and BEBT-908 on
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cellular proliferation in a panel of 5 DLBCL cell lines. All three drugs decreased the viability of lymphoma
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cell in a time- and dose-dependent manner. BEBT-908 displayed potent antiproliferative activity (average IC50 = 0.025 µM, 0.013 µM and 0.009 µM for 24 hours, 48 hours and 72 hours, respectively) in all tested DLBCL cell lines. Notably, BEBT-908 was more potent than SAHA (average IC50 = 1.471 µM, 0.830 µM and 0.754 µM for 24 hours, 48 hours and 72 hours, respectively) and CAL-101 (average IC50 = 22.815
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µM, 12.224 µM and 6.339 µM for 24 hours, 48 hours and 72 hours, respectively) (Table 1). At the 72-hour timepoint, the average IC50 values for the individual lymphoma sub-groups were 0.010 µM (0.008–0.011 µM) for ABC DLBCL cell line and 0.009 µM (0.003–0.019 µM) for GCB DLBCL cell lines (Fig. 1B).
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cells.
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BEBT-908 exhibited no significant difference in growth inhibition between the two sub-groups of DLBCL
To ensure that the antiproliferative activity of BEBT-908 was not because of general cytotoxicity, we tested its growth inhibition effect on PBMCs. The results showed that PBMCs were considerably resistant to the antiproliferative effect of BEBT-908 (IC50 = 23.274 µM) (Table 1). Thus, BEBT-908 exerts potent antiproliferative effect specifically on DLBCL cells.
Table 1 Growth inhibition of DLBCL cell lines by BEBT-908 and reference compounds 10
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CAL-101 (µM)
BEBT-908 (µM)
Cell line 48h
72h
24h
48h
72h
24h
48h
72h
PBMC
-
-
315.13
-
-
890.908
-
-
23.274
TMD8
1.801
0.966
0.742
12.531
2.277
1.198
0.024
0.011
0.008
SU-DHL-2
1.702
0.784
0.809
43.559
27.593
9.852
SU-DHL-6
2.091
1.385
1.258
34.285
14.317
7.959
OCI-LY-7
1.386
0.774
0.674
22.434
16.737
12.607
Pfeiffer
0.373
0.239
0.289
1.266
0.196
0.014
0.011
0.047
0.027
0.019
0.017
0.008
0.006
0.009
0.004
0.003
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0.027
0.079
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PBMC: peripheral blood mononuclear cell
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24h
3.2 BEBT-908 is a potent inhibitor of both PI3K and HDAC and abrogates key DLBCL signaling pathways
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via HDAC inhibition
We conducted Western blot analysis to detect the expression level of p-AKT in the five DLBCL cell lines. Among the cell lines, the highest level of p-AKT was observed in SU-DHL-2 and Pfeiffer cells
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corresponding to ABC- and GCB- DLBCL cells, respectively (Fig. 2A). Based on the results of growth
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inhibitory effects of BEBT-908 and p-AKT expression level in the cell lines, SU-DHL-2 and Pfeiffer cells were selected for subsequent experiments. To confirm that BEBT-908 deactivates PI3K and HDAC in vitro, we further examined its effects in SU-DHL-2 and Pfeiffer cell lines. BEBT-908 inhibited p-AKT expression in a dose-dependent manner, and its inhibitory effect was clearly superior to that of CAL-101 (Fig. 2B and C). An increase in the level of Ac-H3 was also detected in a dose-dependent manner in response to BEBT-908 treatment. Notably, BEBT-908 was more potent than SAHA at deactivating HDAC (Fig. 2D and E). Therefore, BEBT-908 is a 11
ACCEPTED MANUSCRIPT potent inhibitor of both PI3K and HDAC. To investigate whether BEBT-908 suppresses other growth and survival pathways via feedback mechanisms, SU-DHL-2 and Pfeiffer cells were treated with SAHA, CAL-101 or BEBT-908. In ABC-
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(e.g., SU-DHL-2) and GCB- DLBCL cells (e.g., Pfeiffer), the JAK/STAT3 and MAPK/ERK pathway were activated, which resulted in resistance of these cells to CAL-101. In contrast, BEBT-908 and SAHA inhibited the p-STAT3 and p-ERK1/2 in these lymphoma cells (Fig. 2F and G). Therefore, HDAC
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inhibition by BEBT-908 effectively downregulates the JAK/STAT3 and MAPK/ERK pathways.
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The NF-κB pathway is a key pathway in ABC DLBCL cells. We found that BEBT-908 decreased the levels of nuclear p65 but increased the levels of cytoplasmic p65 and IκBα in SU-DHL-2 (Fig. 2F), thereby inhibiting the NF-κB signaling pathway. Therefore, BEBT-908 abrogates multiple signaling pathways
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likely via HDAC inhibition.
3.3 BEBT-908 induces apoptosis in DLBCL cells
BEBT-908 was shown to induce cell death, promote cell cycle arrest or both. Therefore, we used
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Annexin- V/PI staining to investigate the effects of BEBT-908 on apoptosis in DLBCL cells. BEBT-908
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increased the proportion of apoptotic cells, including early and late apoptotic cells in a dose-dependent manner. In addition, it was more potent than SAHA and CAL-101 at the same dose (Fig. 3A and B). While there were approximately 5.00% and 5.68% apoptotic cells in untreated SU-DHL-2 and Pfeiffer cell lines, respectively, the percentage of apoptotic cells increased to 40.34% and 46.45% in these respective cell lines in response to 100 nM BEBT-908 (Fig. 3B). To elucidate the possible mechanism by which BEBT-908 induced apoptosis, we performed Western blot analysis of apoptotic markers. The increase in apoptosis induction in SU-DHL-2 and Pfeiffer cells following treatment with increasing doses of 12
ACCEPTED MANUSCRIPT BEBT-908 was confirmed via analysis of PARP cleavage and Bcl-2 reduction. Moreover, BEBT-908 increased PARP cleavage and decreased Bcl-2 levels to a greater extent than the same concentration of SAHA and CAL-101 did (Fig. 3C). Thus, BEBT-908 likely induces apoptosis in DLBCL cells by
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activating mitochondrial-mediated pathways.
3.4 BEBT-908 arrests the cell cycle at G1 phase in DLBCL cells
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Next, we studied the effect of BEBT-908 on cell cycle progression of DLBCL cells using PI staining.
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Consistent with earlier reports with other HDAC inhibitors[22], we observed that BEBT-908 and SAHA induced cell cycle arrest at G1 phase. In contrast, the same concentration of CAL-101 had no significant effect on the cell cycle. In addition, the proportion of cells arrested at the G1 phase increased with the dose of BEBT-908 (Fig. 4A and B). In control groups of SU-DHL-2 and Pfeiffer cells, only 22.94% and 29.16%
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cells were in G1 phase, respectively, whereas in 100 nM BEBT-908-treated groups of SU-DHL-2 and Pfeiffer cells, the proportion of cells at the G1 phase significantly increased to 66.21% and 66.54%, respectively. Furthermore, while 58.46% and 53.63% of cells were in S phase in the control groups of
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SU-DHL-2 and Pfeiffer cells, respectively, and treatment with 100 nM BEBT-908 reduced the proportion
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to 22.17% and 20.96%, respectively (Fig. 4B). The increase in the proportion of cells in the G1 phase and the decrease in the proportion of cells in S phase in response to BEBT-908 were consistent with the dose-dependent induction of p21 expression. In addition, BEBT-908 induced p21 expression more potently than did SAHA and CAL-101, which was consistent with the flow cytometry results. (Fig. 4C). Therefore, BEBT-908 exerts its inhibitory effects in different DLBCL cell lines (Table 1 and Fig. 1B, CellTiter-Glo® assay) by both inducing apoptosis and promoting cell cycle arrest.
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Discussion
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At present, a growing number of PI3K inhibitors and HDAC inhibitors are being widely tested in
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clinical trials or have been approved for use in the clinic. These compounds show promising activities in indolent BCL and TCL, although they have limited efficacy on DLBCL. A phase II clinical trial of a combination of idelalisib and entospletinib for the treatment of relapsed and refractory NHL demonstrated only 17% ORR in DLBCL[23]. A phase II trial of vorinostat in relapsed DLBCL resulted in only 5.6%
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ORR[24]. Another phase II clinical trial with a pan-HDAC inhibitor panobinostat in relapsed DLBCL displayed 29% ORR[25]. Despite these outcomes, the combination of PI3K/mTOR inhibitor BEZ235 and the HDAC inhibitor panobinostat was found to synergistically promote cell death in DLBCL irrespective
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of histological types[26]. In addition, the simultaneous inhibition of the PI3K/mTOR signaling pathway
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and HDAC with rapamycin and panobinostat in DLBCL cells resulted in growth inhibition and apoptosis of tumor cells[27]. Based on this evidence, we demonstrated that BEBT-908, a novel dual PI3K/HDAC inhibitor, exerts a potent activity against diverse DLBCL cell lines in vitro. In this study, we found that the administration of very low (nanomolar) concentrations of BEBT-908 suppressed cell proliferation, inhibited AKT phosphorylation and increased histone H3 acetylation in ABCand GCB- DLBCL cell lines but not in normal PBMCs, highlighting the potential therapeutic selectivity of BEBT-908. Notably, the level of AKT phosphorylation was independent of the sensitivity of DLBCL cell 14
ACCEPTED MANUSCRIPT lines to BEBT-908. Thus, AKT phosphorylation may not be the only determining factor of the responsiveness of DLBCL cells to BEBT-908, although a high basal p-AKT level might indicate an increase in the dependence of cells on the PI3K/AKT pathway for survival. BEBT-908 induced apoptosis,
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PARP cleavage, Bcl-2 downregulation, G1 cell cycle arrest and p21 accumulation in DLBCL cells. HDAC inhibitors typically induce G1 cell cycle arrest through the upregulation of the cyclin-dependent kinase inhibitor p21[28]. HDAC inhibitors can also activate the mitochondrial-mediated apoptotic pathway and
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regulate the equilibrium between pro- and anti-apoptotic proteins[22,29]. BEBT-908 was more potent than
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SAHA and CAL-101. The low efficacy of SAHA and CAL-101 might explain the poor outcomes of DLBCL patients in response to these agents in clinical trials.
We also found that BEBT-908 inhibited multiple signaling pathways associated with DLBCL oncogenesis. Apart from suppressing the activation of its direct targets, namely PI3K and HDAC,
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BEBT-908 also effectively reduced the activation of JAK/STAT3, MAPK/ERK, and NF-κB, none of which are the direct targets of BEBT-908. Similar effects were observed in response to SAHA. HDAC inhibitors lead to the acetylation of histone and non-histone protein, which subsequently regulate the activity or
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expression of functional proteins, including STAT3, NF-κB, HIF-1α, c-Myc, p53[30,31].
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Therefore, the synchronous effect on multiple targets, such as PI3K and HDAC, may be a more efficient approach than acting on single targets. The major advantages of a single multi-targeted inhibitor over the combination of several single targeted drugs are simplified pharmacokinetics, easy administration, reduced side effects and improved patient compliance. In addition, it is more effective to combine a single multi-targeted inhibitor with other novel small molecule inhibitors or chemotherapeutic drugs for further clinical development. In conclusion, we have discovered a highly selective and potent dual PI3K/HDAC inhibitor, BEBT-908, 15
ACCEPTED MANUSCRIPT which displays better antiproliferative effects against DLBCL cells than single targeted PI3K and HDAC inhibitors. BEBT-908 thus represents a novel and alternative approach to enhance the activity of PI3K and HDAC inhibitors, and it warrants further clinical investigation to be validated as a therapeutic agent for
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DLBCL.
Acknowledgements
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None.
Funding
This work was supported by the Guangzhou BeBetter Medicine Technology Co., Ltd.
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Conflict of interest
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There are no potential conflicts of interest to disclose
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Figure 1. Growth inhibition of DLBCL cell lines by BEBT-908. A, Structure of BEBT-908. B, IC50 curves
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in 5 DLBCL cell lines after 24 hours, 48 hours and 72 hours of BEBT-908 exposure.
Figure 2. Inhibition of PI3K, HDAC and multiple signaling pathways by BEBT-908. SU-DHL-2 and Pfeiffer cells were treated with DMSO, 100 nM SAHA, 100 nM CAL-101, and 100 nM BEBT-908 or with
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DMSO and increasing concentrations of BEBT-908 for 24 hours. A, Expression of p-AKT in 5 DLBCL cell lines. B and C, Inhibition of p-AKT. D and E, Increase of Ac-H3. F, Inhibition of JAK/STAT3,
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in Pfeiffer.
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MAPK/ERK and NF-κB pathways in SU-DHL-2. G, Inhibition of JAK/STAT3 and MAPK/ERK pathways
Figure 3. BEBT-908 induces apoptosis. SU-DHL-2 and Pfeiffer cells were treated with DMSO, 100 nM SAHA, 100 nM CAL-101, and 100 nM BEBT-908 or with DMSO and increasing concentrations of BEBT-908 for 24 hours. A, Representative histograms of Annexin-V /PI staining assessed using flow cytometry. B, Percentage of apoptotic cells (including early and late apoptotic cells) determined using flow cytometry. *p<0.05 vs control. C, Western blot analysis for apoptotic markers in cell lysates.
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ACCEPTED MANUSCRIPT Figure 4. BEBT-908 induces G1 phase cell cycle arrest. SU-DHL-2 and Pfeiffer cells were treated with DMSO, 100 nM SAHA, 100 nM CAL-101, and 100 nM BEBT-908 or with DMSO and increasing concentrations of BEBT-908 for 24 hours. Flow cytometry analysis using PI staining to assess cell cycle
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distribution. A, Representative histograms of cell cycle distribution assessed by PI staining. B, Bar graphs depicting the relative percentage of cells in each phase of the cell cycle in response to BEBT-908 and
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reference compounds. *p<0.05 vs control. C, Western blot analysis of p21 expression.
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ACCEPTED MANUSCRIPT Highlights 1. BEBT-908 exerts cytotoxic and antiproliferative effects on DLBCL. 2. BEBT-908 represses the PI3K/AKT pathway and HDAC activity in DLBCL cells.
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4. BEBT-908 induces apoptosis and G1 phase arrest in DLBCL cells.
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3. BEBT-908 inhibits key DLBCL pathways, including JAK/STAT3, MAPK/ERK and NF-κB.