progenitor cell function

Accepted Manuscript Targeting of the BLT2 in chronic myeloid leukemia inhibits leukemia stem/progenitor cell function Meifang Xiao, Hongmei Ai, Tao Li, Pasupati Rajoria, Prakash Shahu, Xiansong Li PII:

S0006-291X(16)30327-8

DOI:

10.1016/j.bbrc.2016.03.018

Reference:

YBBRC 35450

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 29 February 2016 Accepted Date: 6 March 2016

Please cite this article as: M. Xiao, H. Ai, T. Li, P. Rajoria, P. Shahu, X. Li, Targeting of the BLT2 in chronic myeloid leukemia inhibits leukemia stem/progenitor cell function, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.03.018. 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 Targeting of the BLT2 in chronic myeloid leukemia inhibits leukemia stem/progenitor cell function

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Meifang Xiao1, Hongmei Ai1, Tao Li1, Pasupati Rajoria2, Prakash Shahu2, Xiansong Li3*

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University of Science and Technology (HUST ), Jing Zhou, People's Republic of China

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Republic of China

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Department of Laboratory Medicine, JingZhou Hospital, Tongji Medical College, Huazhong

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Department of Clinical Medicine, Medical School of Yangtze University, Jingzhou, People's

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University of Science and Technology (HUST ), Jing Zhou, People's Republic of China

Department of Neurosurgery, JingZhou Hospital, Tongji Medical College, Huazhong

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*Corresponding author: Xiansong Li, Department of Neurosurgery, JingZhou Hospital,

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Tongji Medical College, Huazhong University of Science and Technology (HUST), Renmin

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Road 1, Jing Zhou, People's Republic of China 434020;

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Email address: [email protected]

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ACCEPTED MANUSCRIPT Abstract

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Imatinib, a tyrosine kinase inhibitor (TKI) has significantly improved clinical outcome for

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chronic myeloid leukemia (CML) patients. However, patients develop resistance when the

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disease progresses to the blast phase (BP) and the mechanisms are not well understood. Here

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we show that BCR-ABL activates BLT2 in hematopoietic stem/progenitor cells to promote

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leukemogenesis and this involves the p53 signaling pathway. Compared to normal bone

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marrow (NBM), the mRNA and protein levels of BLT2 are significantly increased in BP-

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CML CD34+ stem/progenitor cells. This is correlated with increasing BCR-ABL expression.

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In contrast, knockdown of BCR-ABL or inhibition of its tyrosine kinase activity decreases

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Blt2 protein level. BLT2 inhibition induces apoptosis, inhibits proliferation, colony formation

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and self-renewal capacity of CD34+ cells from TKI-resistant BP-CML patients. Importantly,

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the inhibitory effects of BCR-ABL TKI on CML stem/progenitor cells are further enhanced

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upon combination with BLT2 inhibition. We further show that BLT2 activation selectively

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suppresses p53 but not Wnt or BMP-mediated luciferase activity and transcription. Our

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results demonstrate that BLT2 is a novel pathway activated by BCR-ABL and critically

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involved in the resistance of BP-CML CD34+ stem/progenitors to TKIs treatment. Our

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findings suggest that BLT2 and p53 can serve as therapeutic targets for CML treatment.

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Key words: Chronic myeloid leukemia, BLT2, p53, Bcr-Abl tyrosine kinase inhibitor

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ACCEPTED MANUSCRIPT 1. Introduction

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Chronic myeloid leukemia (CML) is a hematological stem cell malignancy resulting from the

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transformation by oncogene BCR-ABL [1]. BCR-ABL activates proliferation and survival

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pathways in hematopoietic stem/progenitor cells. Therefore, treatment with the BCR-ABL

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tyrosine kinase inhibitors (TKIs), such as imatinib, has emerged as the first-line treatment for

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CML patients. However, when CML progresses to blast phase (BP), imatinib is not effective

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in eliminating residual leukemia progenitor cells that may serve as a source for relapse [2, 3].

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The mechanisms underlying resistance to TKIs are not well understood and may involve

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BCR-ABL-independent mechanisms [4].

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Leukotriene B4 receptor 2 (BLT2) is a receptor of leukotriene B4 and 12 (S)-hydroxyhe-

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ptadeca-5Z, 8E, 10E-trienoic acid (12-HHT) [5, 6]. BLT2 has been shown to play essential

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roles in cancer progression. It is up-regulated in a variety of human cancers and mediates

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Ras-induced transformation [7, 8]. Treatment with BLT2-specific antagonist LY255283

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induces cell cycle arrest and apoptosis in cancer cells derived from various origins including

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prostate, bladder and breast [8-10]. BLT2 also enhances cancer invasion and metastasis by

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up-regulating matrix metalloproteinase-9 [11]. Recently, it has been reported that the

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arachidonic acid–LTB4–BLT2 pathway enhances human chronic lymphocytic leukemia (B-

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CLL) aggressiveness [12]. However, little is known about the role of BLT2 in CML.

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In this study, we have investigated the role of BCR-ABL in the expression of BLT2 in

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normal bone marrow (NBM) and BP-CML CD34+ stem/progenitor cells. Our results show

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that BCR-ABL regulates BLT2 expression to promote leukemogenesis and this involves

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inhibition of p53 signaling pathway. We also demonstrate the essential roles of BLT2 in

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growth, differentiation, self-renewal and survival of CML CD34+ stem/progenitor cells.

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ACCEPTED MANUSCRIPT Finally, we show that the concurrent inhibition of BLT2 and BCR-ABL are synergistic in

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eliminating BP-CML CD34+ cells.

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2. Materials and methods

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2.1 Patient samples and retroviral transduction of human Normal bone marrow (NBM)

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CD34+ cells

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CD34+ cells were purified using CD34 MicroBead kit (Miltenyi Biotec, Germany) from bone

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marrows mononuclear cells of BP-CML patients seen at JingZhou Hospital, Tongji Medical

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College, Huazhong University of Science and Technology (HUST). Written informed

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consents were obtained from all patients under protocols approved by the institutional review

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board. NBM CD34+ cells were purchased from StemCell Technologies, Inc. BP-CML and

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normal bone marrow CD34+ cells were cultured in serum-free StemPro complete medium

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(Life Technologies, US) supplemented with stem cell factor, 0.2 ng/mL; granulocyte-

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macrophage colony-stimulating factor, 0.2 ng/mL; macrophage inflammatory protein-1α, 0.2

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ng/mL; granulocyte colony−stimulating factor, 1 ng/mL; leukemia inhibitory factor, 50

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pg/mL; and interleukin 6, 1 ng/mL)[13]. NBM CD34+ cells were transduced using retroviral

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vectors NGFR and NGFR P210 (kind gifts from Dr. Warren Pear) as described previously

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[14]. Cells were harvested 48 hours later and CD34+GFP+ cells were collected by flow

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cytometry (Beckman Coulter FC500, Beckman Coulter, France).

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2.2 Cell culture, Generation of cell lines, siRNA transfection and Chemicals

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Human CML cell line KCL22 was obtained from American Type Culture Collection (ATCC)

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and cultured in RPMI1640 medium (Life Technologies, CA, US) supplemented with 10%

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fetal bovine serum (FBS) (Hyclone, UK). KCL22 (BLT2) line overexpressing BLT2 were

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generated by stable transfection using pcDNA3-BLT2 plasmid (a kind gift from Dr. Shimizu)

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ACCEPTED MANUSCRIPT as previously described [15]. Selected KCL22 (BLT2) colonies were maintained in the

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presence of 0.4 mg/ml of G418 (Invitrogen, US). BLT2 or Abl knockdown were carried out

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in KCL 22 cells by transfecting with 100 nM scramble siRNA (SCR) or human BLT2 or Abl-

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specific siRNAs using Dharmafect Transfection Reagent (Dharmacon RNAi Technologies).

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The target sequence of human Abl-specific is siABL: GCA GAG UUC AAA AGC CCU

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UdT dT [16]. The target sequences of human BLT2-specific are siRNA BLT2a: AAC ATC

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GCC CTG TGG ATG ACT, siRNA BLT2b: AGT ACA TCC ATT ATA AGC T. Dasatinib

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and imatinib were purchased from LC laboratories. LY255283 was purchased from Tocris

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Bioscience.

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2.3 Western blotting

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Whole protein from cells were lysed by RIPA lysis buffer (Life Technologies Inc, US), then

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processed for western blot analysis by using antibodies anti-β-catenin, anti-p- β-catenin

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(S552) (BD Transduction Labs, US), anti-Bcr-Abl, anti- BLT2, anti-p-Crkl (Santa Cruz

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Biotechnology, US), anti-Ac-p53 (K382), anti-p53, anti-SMAD1/5, anti-p-SMAD1/5, anti-p-

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MAPK, anti-MAPK and anti-actin (Cell Signaling Technologies, US).

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2.4 Colony-forming and serial replating assays

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CD34+ BP-CML cells were subjected to drug treatment for 48 hours. Equal number of live

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cells were then seeded in HSC-CFU methylcellulose medium (Miltenyi Biotec, Germany).

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For colony-forming assay, the number of colonies was scored after 14 days. For serial

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replating assays, individual colonies formed in colony-forming assay were picked and

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replated in HSC-CFU complete methylcellulose in a 96-well format, and counted at 14 days.

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Three rounds of serial replating representing more than 8 weeks in culture were performed.

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Serial replating capacity is determined by the percentage of final number of positive wells

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among total number of colonies plated.

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ACCEPTED MANUSCRIPT 2.5 Measurement of proliferation and apoptosis

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Cells were treated with drug alone or combination for 72 hours. Proliferation activity was

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measured by the CellTiter 96R AQueous One Solution Cell Proliferation assay kit (Promega,

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US). For apoptosis, cells were stained with Annexin V-FITC (Beckman Coulter, France) and

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the percentage of Annexin V-positive cells was analysed using flow cytometry.

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2.6 Real-Time Q-PCR analysis

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The total RNA was isolated by using TRIzol Reagent (Life technologies, CA, US). Q-PCR

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analysis performed by using a SsoFast EvaGreen Supermix and CFX96 RT PCR system

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(Bio-rad, CA). The primers are for human BCL-2 (5’-CTG CAC CTG ACG CCC TTC ACC-

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3’and 5’-CAC ATG ACC CCA CCG AAC TCA AAG A-3’), BCR-ABL (5'-ACT TGT CGT

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AGT TGG GGG ACA CAC CA-3' and 5'-GCG AAC AAG GGC AGC AAG GCT ACG-3’ ),

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P21 (5’-GAG GCC GGG ATG AGT TGG GAG GAG-3’ and 5’-CAG CCG GCG TTT GGA

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GTG GTA GAA-3’), NDN (5’- GGT CCC CGA CTG TGA GAT GC-3’ and 5’-CGA GGT

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TAG CGG CCA GAG AA-3’) and PUMA (5’-ACC TCA ACG CAC AGT ACG AG-3’ and

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5’-CCC ATG ATG AGA TTG TAC AGG A-3’) and β-actin (5’-AAG GAT TCC TAT GTG

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GGC GAC G-3’ and 5’-GCC TGG ATA GCA ACG TAC ATG G-3’).

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2.7 Reporter assays

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Transfections were carried out in KCL22 (Vec) and KCL22 (BLT2) cells by using

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nucleofection (Lonza). Cells were transfected with M50 Super 8x TOPFlash (a kind gift

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from Dr. Randall Moon) [17], PG13-luc (wt p53 binding sites, a kind gift from Dr. Bert

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Vogelstein) [18], or pGL3 BRE plasmid (a kind gift from Dr. Martine Roussel & Peter ten

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Dijke) [19] to assess the effects of BLT2 on Wnt, p53, or BMP transcriptional activity,

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respectively.

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ACCEPTED MANUSCRIPT 2.8 Statistical analyses

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All data are expressed as mean ± SEM. Student’s t test was performed to determine statistical

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significance.

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3. Results

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3.1 BLT2 is activated by BCR-ABL transformation.

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To determine whether BCR-ABL expression regulates BLT2 in CML, we isolated normal

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bone marrow (NBM) and BP-CML CD34+ stem/ progenitor cells and investigated the

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expression levels of BLT2 and BCR-ABL. We found that both mRNA and protein expression

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levels of BLT2 were low in NBM CD34+ progenitor cells, but were significantly increased in

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BP-CML progenitor cells (Fig. 1A and B). We next determined the change of BLT2

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expression during BCR-ABL transformation. NBM CD34+ cells were transduced with the

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BCR-ABL retroviral vector NGFR P210 [14]. Consistently, the BLT2 mRNA and protein

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level were up-regulated by BCR-ABL transduction (Fig.1C and D). We further found that

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knockdown BCR-ABL decreased Blt-2 protein level (Fig. 1E). Similarly, when the tyrosine

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kinase activity was suppressed by BCR-ABL inhibitor imatinib as shown by the decreased p-

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Crkl (a marker of BCR-ABL activity) in CML KCL22 cells, Blt2 expression was decreased

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in a drug concentration-dependent manner (Fig.1F). These data support that BCR-ABL

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activates BLT2 expression in CML cells.

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3.2 BLT2 inhibition by genetic and pharmacological approaches suppresses growth and

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induces apoptosis of CML cells.

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To determine the consequence of BLT2 activation, we first depleted BLT2 in KCL22 cells

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with two independent BLT2 siRNA (Fig. 2A). We observed that depletion of BLT2 led to

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decreased growth and increased apoptosis compared to control cells (Fig.2 B and C).

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ACCEPTED MANUSCRIPT Consistently, we treated KCL22 cells with LY255283, a selective and competitive antagonist

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of BLT2 receptor [20] and observed that LY255283 inhibited proliferation and induced

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apoptosis of KCL22 cells in a dose-dependent manner (Fig. 2D and E). These data

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demonstrate that BLT2 is important for CML cell growth and survival.

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3.3 BLT2 inhibitor alone, and synergistically with BCR-ABL TKI, induces apoptosis, inhibits

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colony formation and self-renewal of BP-CML CD34+ stem/progenitor cells.

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Compared to chronic phase CML, BCR-ABLTKIs are less effective as single agents in BP-

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CML cells. We therefore investigated whether BLT2 inhibition is effective in CD34+

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stem/progenitor cells derived from BP-CML patients and its combination with BCR-

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ABLTKIs resulted in greater efficacy than each single drug. Consistent with CML cell line

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results, LY255283 induced dose-dependent apoptosis in CD34+ cells in BP-CML patients.

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The combination of LY255283 and dasatinib (2nd generation TKIs for BP-CML treatment)

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further enhanced apoptosis compared to the single agent alone (Fig. 3A).

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The hallmark characteristics of stem/progenitor cells are to self-renew, proliferate and

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differentiate [21]. To test whether BLT2 inhibition also affects proliferation, differentiation

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and self-renewal of BP-CML CD34+ cells, we performed colony-forming and serial replating

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assays. We observed that LY255283 decreased colony formation, and its combination with

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dasatinib completely abolished colony formation of BP-CML CD34+ cells (Fig.3B and C).

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We also observed that DMSO-treated BP-CML CD34+ cells were able to maintain their

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replating efficiency to a third replating. However, exposure to LY255283 significantly

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impaired the ability of these cells to serially replate (Fig. 3D). Similarly, the combination of

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LY255283 and dasatinib completely abolished the self-renewal ability during the third

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replating (Fig. 3D). Altogether, our findings demonstrate that BLT2 inhibition is able to

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induce apoptosis, prevent colony formation and serial replating functions of CML

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ACCEPTED MANUSCRIPT stem/progenitor cells. In addition, BLT2 inhibition further enhanced the effects of BCR-ABL

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TKI in eliminating CML stem/progenitor cells.

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3.4 Activation of BLT2 suppresses p53 signaling pathway in CML cells.

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To understand the mechanisms of BLT2 activation in CML cells, we investigated the

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important pathways regulating functions of CML stem/progenitor cells including β-catenin

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pathway, p53 pathway and BMP pathway [22-24]. We generated KCL22 (BLT2) line that

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stably overexpresses BLT2. We observed the increased Bcr-Abl activities in KCL22 (BLT2)

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cells as shown by the increased phosphorylation of Crkl and mitogen-activated protein kinase

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(MAPK) (Fig. 4A).

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Importantly, we observed the decreased acetylated p53 levels and no discernible differences

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on the phosphorylation of β-catenin or SMAD1/5 (Fig. 4A). These results demonstrate that

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BLT2 overexpression is likely to inhibit p53 but not Wnt or BMP signaling pathways. This is

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confirmed by the data that p53 but not Wnt or BMP-mediated luciferase activity is decreased

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in KCL22 (BLT2) compared to control cells (Fig. 4B). Consistent with the inhibition of p53

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signalling, the mRNA expression levels of p53 target genes including p21, Ndn, Puma and

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Bax were decreased in KCL22 (BLT2) cells (Fig. 4C). Altogether, our data demonstrate the

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specificity of BLT2 overexpression for p53 signaling pathway.

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4. Discussion

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Although recent studies highlighted the important role of BLT2 in cancer cell survival and

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maintenance [8-11], little is known whether BLT2 expression and activation is required for

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leukemogenesis, especially in primary leukemia CD34+ stem/progenitor population. Using

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the CML disease model, we are the first to demonstrate that BLT2 activation by BCR-ABL

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ACCEPTED MANUSCRIPT tyrosine kinase is essential to promote leukemogenesis through activating p53 signaling

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pathway and targeting BLT2 represents a potential therapeutic strategy for CML treatment.

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The BLT2 mRNA and protein levels are correlated with increasing BCR-ABL expression in

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CD34+ stem/ progenitor cells derived from CML patients (Fig. 1A and B). Consistently, the

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BLT2 mRNA and protein level are up-regulated by BCR-ABL transduction in NBM

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progenitor cells (Fig.1C and D). This is direct evidence obtained from primary leukemia and

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normal hematopoietic progenitor populations, and suggests that BLT2 is activated by BCR-

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ABL transformation. This conclusion is further supported by our data that knockdown BCR-

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ABL or inhibition of its tyrosine kinase activity decreased Blt2 protein level (Fig. 1E and F).

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BLT2 is involved in a wide array of cytokine signaling for cancer growth, survival and

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invasiveness [8, 12, 25]. In agreement with this notion, our finding that BLT2 activation by

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oncogene BCR-ABL transformation suggests that BLT2 activation may serve as a common

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signaling pathway in response to oncogenic stress in cancer.

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A role of BLT2 activation by BCR-ABL is to maintain CML cell growth and survival as

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demonstrated by the decreased growth and increased apoptosis in BLT2-depleted CML cells

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and cells treated with selective BLT2 inhibitor LY255283 (Fig. 2). These data add to the

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recent evidence supporting the important role of BLT2 in the growth and survival of cancer

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[9, 26]. Our work further demonstrate that targeting BLT2 is also effective in eliminating

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CD34+ stem/progenitor cells derived from CML patients via inducing apoptosis, inhibiting

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colony formation and self-renewal capability (Fig. 3). BLT2 has been reported to contribute

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chemoresistance of SK-OV-3 ovarian cancer cells [27]. This is supported by our data that

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LY255283 acts synergistically with 2nd generation BCR-ABL TKI dasatinib on CML CD34+

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stem/progenitor cells (Fig.3). Our study suggests the therapeutic value of targeting BLT2 in

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overcoming resistance to BCR-ABL TKIs in CML.

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ACCEPTED MANUSCRIPT Activation of BLT2 are associated with activation of ERK/AKT, JAK/STAT3. JNK/p38 or

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NF-ĸB in the progression of different cancer [8]. Our study is the first to add p53 to the

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growing list of BLT2-regulated downstream signaling. In CML cells that stably overexpress

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BLT 2, we observed the increased Bcr-Abl activities as shown by the increased

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phosphorylation of Crkl and MAPK, followed by the decreased level of acetylated p53,

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decreased p53-mediated luciferase activity and decreased mRNA levels of p53 target genes

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(Fig. 4). In contrast, we neither observed differences on the phosphorylation of Wnt and BMP

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effectors (eg. β-catenin and SMAD1/5), nor Wnt or BMP-mediated luciferase activity (Fig.

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4A and B), thereby demonstrating specificity of BLT2 activation for p53 signaling pathway.

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Collectively, these data suggest that BLT2 activation promotes leukemogenesis through

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suppressing p53 signaling pathway. This finding supports the previous study that activation

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of p53 enhances elimination of CML cells in combination with imatinib [23].

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In conclusion, our work on the identification of BLT2 activation by BCR-ABL as an essential

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step in leuekogenesis expands the previously known BCR-ABL-regulated molecular network

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that controls CML cell functions. This work also provides better understanding of the

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molecular mechanisms underlying persistence of leukemia stem/progenitor cells in TKI-

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treated CML patients and suggests a rationale for targeting BLT2 and p53 to overcome this

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resistance.

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Conflict of interest

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All authors declare no conflict of interest.

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Acknowledgement

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We thank Dr. Randall Moon for his kind gift of M50 Super 8x TOPFlash, Dr. Bert

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Vogelsteinf for his kind gift of PG13-luc, Dr. Nicholas Gaiano for his kind gift of pCBFRE-

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ACCEPTED MANUSCRIPT (mt)-luc, Dr. Martine Roussel & Peter ten Dijke for their kind gift of pGL3 BRE, Dr. Shimizu

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for his kind gift of BLT2/pcDNA3 plasmid. This work was supported by a research grant

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provided by JingZhou Hospital, Tongji Medical College, Huazhong University of Science

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and Technology (HUST) (201306016).

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References

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ACCEPTED MANUSCRIPT [15] T. Yokomizo, K. Kato, K. Terawaki, T. Izumi, T. Shimizu, A second leukotriene B(4) receptor, BLT2. A new therapeutic target in inflammation and immunological disorders, J Exp Med, 192 (2000) 421-432. [16] M. Scherr, K. Battmer, T. Winkler, O. Heidenreich, A. Ganser, M. Eder, Specific inhibition of bcr-abl gene expression by small interfering RNA, Blood, 101 (2003) 15661569. [17] M.T. Veeman, D.C. Slusarski, A. Kaykas, S.H. Louie, R.T. Moon, Zebrafish prickle, a modulator of noncanonical Wnt/Fz signaling, regulates gastrulation movements, Curr Biol, 13 (2003) 680-685. [18] W.S. el-Deiry, T. Tokino, V.E. Velculescu, D.B. Levy, R. Parsons, J.M. Trent, D. Lin, W.E. Mercer, K.W. Kinzler, B. Vogelstein, WAF1, a potential mediator of p53 tumor suppression, Cell, 75 (1993) 817-825. [19] O. Korchynskyi, P. ten Dijke, Identification and functional characterization of distinct critically important bone morphogenetic protein-specific response elements in the Id1 promoter, J Biol Chem, 277 (2002) 4883-4891. [20] D.K. Herron, T. Goodson, N.G. Bollinger, D. Swanson-Bean, I.G. Wright, G.S. Staten, A.R. Thompson, L.L. Froelich, W.T. Jackson, Leukotriene B4 receptor antagonists: the LY255283 series of hydroxyacetophenones, J Med Chem, 35 (1992) 1818-1828. [21] S. Takeishi, K.I. Nakayama, Role of Fbxw7 in the maintenance of normal stem cells and cancer-initiating cells, Br J Cancer, (2014). [22] C.H.M. Jamieson, L.E. Ailles, S.J. Dylla, M. Muijtjens, C. Jones, J.L. Zehnder, J. Gotlib, K. Li, M.G. Manz, A. Keating, C.L. Sawyers, I.L. Weissman, Granulocyte-macrophage progenitors as candidate leukemic stem cells in blast-crisis CML, New Engl J Med, 351 (2004) 657-667. [23] L. Li, L. Wang, L. Li, Z. Wang, Y. Ho, T. McDonald, T.L. Holyoake, W. Chen, R. Bhatia, Activation of p53 by SIRT1 inhibition enhances elimination of CML leukemia stem cells in combination with imatinib, Cancer Cell, 21 (2012) 266-281. [24] B. Laperrousaz, S. Jeanpierre, K. Sagorny, T. Voeltzel, S. Ramas, B. Kaniewski, M. Ffrench, S. Salesse, F.E. Nicolini, V. Maguer-Satta, Primitive CML cell expansion relies on abnormal levels of BMPs provided by the niche and on BMPRIb overexpression, Blood, 122 (2013) 3767-3777. [25] J.W. Lee, J.H. Kim, Activation of the leukotriene B4 receptor 2-reactive oxygen species (BLT2-ROS) cascade following detachment confers anoikis resistance in prostate cancer cells, J Biol Chem, 288 (2013) 30054-30063. [26] H. Kim, J.A. Choi, G.S. Park, J.H. Kim, BLT2 up-regulates interleukin-8 production and promotes the invasiveness of breast cancer cells, PLoS One, 7 (2012) e49186. [27] J. Park, S.Y. Park, J.H. Kim, Leukotriene B4 receptor-2 contributes to chemoresistance of SK-OV-3 ovarian cancer cells through activation of signal transducer and activator of transcription-3-linked cascade, Biochim Biophys Acta, 1863 (2016) 236-243.

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Fig. 1. BCR-ABL expression activates BLT-2 in human hematopoietic progenitor cells.

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(A) mRNA and (B) protein levels of BCR-ABL and BLT2 in NBM and BP-CML CD34+

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cells. (C) mRNA and (D) protein levels of BCR-ABL and BLT2 after BCR-ABL

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transduction (NGFR P210) in NBM CD34+ cells. (E) BLT2 expression on BCR-ABL

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nM SCR or siABL. SCR indicates scrambled siRNA. (F) Change of Blt2 protein levels on

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Imatinib treatment for 2 days in KCL22 cells.

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Fig. 2. BLT2 inhibition inhibits proliferation and induces apoptosis of KCL22 cells. (A)

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Blt2 protein level on BLT2 knockdown by two independent siRNA in KCL22 cells. (B)

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Proliferation is decreased and (C) apoptosis is increased in Blt2 depleted KCL22 cells. Cells

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are electroporated with 100 nM SCR or siRNA Blt2 (#a or #b) and cultured for 24 hours prior

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to assays. LY255283 inhibits proliferation (D) and induces apoptosis (E) of KCL22 cells.

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Fig. 3. BLT2 inhibitor alone, and acts synergistically with dasatinib in CML CD34+ cells.

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(A) LY255283 induces apoptosis of BP-CML CD34+ cells and combination of LY255283

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and dasatinib induces more apoptosis than single drug alone. (B) Representative images taken

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at 14 days of a colony-forming assay. LY255283 significantly reduces colony formation (C)

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and serial replating capacity (D) of CML and enhances the inhibitory effects of dasatinib.

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Colonies were enumerated and individually picked for serial replating. Graphs presented are

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mean of the results obtained from twenty BP-CML patients. *p<0.05, compared to single arm

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treatment.

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Fig. 4. Activation of BLT2 suppresses p53 signaling pathway in CML cells. (A) Western

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blots of KCL (Blt2) cells stably overexpress Blt2. Western blot analysis using antibodies

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recognizing Blt2, Ac-p53(K382), p53, Bcr-Abl, p-Crkl, p-β-catenin (S552), β-catenin, p-

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SMAD1/5, SMAD1/5, p-MAPK, MAPK and β-actin. Compared to control KCL (Vec) cells,

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(B) p53 but not Wnt or BMP-mediated luciferase activity and (C) the mRNA expression level

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of p53 target genes are decreased in KCL22 (Blt2) cells. Cells were transfected with

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luciferase-based reporter constructs as described in methods. *p<0.05, compared to control.

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Highlights •

BCR-ABL regulates BLT2 expression to promote leukemogenesis.

• BLT2 is essential to maintain CML cell function. • Activation of BLT2 suppresses p53 signaling pathway in CML cells.

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Inhibition of BLT2 and BCR-ABL synergize in eliminating CML CD34+ stem/progenitors.

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