PBA2, a novel inhibitor of imatinib-resistant BCR-ABL T315I mutation in chronic myeloid leukemia

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54

CAN13048_proof ■ 7 October 2016 ■ 1/10

Cancer Letters xxx (2016) 1e10

Contents lists available at ScienceDirect

Cancer Letters journal homepage: www.elsevier.com/locate/canlet

Original Article

Q6 Q5

PBA2, a novel inhibitor of imatinib-resistant BCR-ABL T315I mutation in chronic myeloid leukemia Pranav Gupta a, Rishil J. Kathawala a, Liuya Wei a, b, Fang Wang c, XiaoKun Wang c, Brian J. Druker d, Li-Wu Fu c, **, Zhe-Sheng Chen a, * a

Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John's University, Queens, NY, 11439, USA School of Pharmacy, Weifang Medical University, Weifang, 261053, China SunYat-Sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou, 510060, China d Division of Hematology and Medical Oncology, The Knight Cancer Institute, Oregon Health and Science University, Portland, OR, 97239, USA b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 August 2016 Received in revised form 6 September 2016 Accepted 7 September 2016

Chronic Myeloid Leukemia (CML) is largely caused by the Philadelphia (Ph) chromosome carrying the Break point Cluster Region-Abelson (BCR-ABL) oncogene. Imatinib is a BCR-ABL-targeted therapy and considered the standard of care in CML management. Resistance to imatinib therapy often develops because of mutations in the BCR-ABL kinase domain. In this study, we evaluated PBA2, a novel BCR-ABL inhibitor, for its anti-cancer activity against BCR-ABL expressing BaF3 cells. PBA2 shows potent activity against wild-type and T315I mutated BaF3 cells as compared with imatinib. PBA2 inhibited the phosphorylation of BCR-ABL and its downstream signaling in BaF3/WT and BaF3/T315I cells. PBA2 inhibited the mRNA expression of BCR-ABL in BaF3/WT and BaF3/T315I cells. Mechanistically, PBA2 increased the cell population in sub G1 phase of the cell cycle, induced apoptosis and elevated ROS production in both BaF3/WT and BaF3/T315I cells. Taken together, our results indicate that PBA2 exhibits anti-proliferative effects and inhibits the imatinib-resistant T315I BCR-ABL mutation. PBA2 may be a novel drug candidate for overcoming the resistance to imatinib in CML patients. © 2016 Elsevier Ireland Ltd. All rights reserved.

Keywords: PBA2 Chronic myeloid leukemia BCR-ABL T315I Resistance

Introduction Chronic myeloid leukemia (CML), a myeloproliferative disorder, is a clonal expansion of the progenitor hematopoietic stem cells [1e4]. The National Cancer Institute (NCI) has defined CML as a disease that slowly progresses in the blood and bone marrow and usually occurs after middle age, that is, between 45 and 55 years [5]. The American Cancer Society has estimated that about 8220 new cases of CML were diagnosed in 2016 in the United States, amongst which, 4610 were men and 3610 were women [6]. Moreover, about 1070 deaths (570 men and 500 women) were estimated from CML [6]. CML accounts for around 10% of all the new

Q1 Q2

* Corresponding author. Fax: þ1 718 990 1877. ** Corresponding author. Cancer Center, Sun Yat-Sen University, Guangzhou, 510060, Guangdong Province, China. Fax: þ86 20 873 431 70. E-mail addresses: [email protected] (L.-W. Fu), [email protected] (Z.-S. Chen).

cases of leukemia, with a greater prevalence in men (ratio of 1.3 to 1) [7]. CML is caused by the formation of Philadelphia chromosome which accounts for 90% of CML cases [8,9]. A reciprocal translocation t(9; 22) and fusion between the ABL tyrosine kinase gene on chromosome 9 and the BCR tyrosine kinase gene on chromosome 22 results in the formation of BCR-ABL oncogene on the Philadelphia chromosome [10]. The BCR-ABL oncogene exists in three different forms (p210, p185, and p230), where each form has distinct structural domains and produces a distinct leukemia type [11,12]. The tumorigenic potential of the BCR-ABL lies in the fact that this fusion gene leads to the production and activation of other signaling molecules [13]. These molecules constitute a pathway that leads to an increase in proliferation and survival of leukemic cells. Interestingly, these signaling pathways play a major role in producing drug resistance to tyrosine kinase inhibitors (TKIs). STI571, now known as imatinib, was the first BCR-ABL TKI approved by the United States Food and Drug Administration for the treatment of CML and gastrointestinal stromal tumor (GIST)

http://dx.doi.org/10.1016/j.canlet.2016.09.025 0304-3835/© 2016 Elsevier Ireland Ltd. All rights reserved.

Please cite this article in press as: P. Gupta, et al., PBA2, a novel inhibitor of imatinib-resistant BCR-ABL T315I mutation in chronic myeloid leukemia, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.09.025

55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

CAN13048_proof ■ 7 October 2016 ■ 2/10

2

P. Gupta et al. / Cancer Letters xxx (2016) 1e10

[14e16]. Although the majority of patients benefited from imatinib treatment, a certain portion became unresponsive over the course of therapy. Imatinib resistance occurred in 15% of patients due to point mutations [17], BCReABL gene amplification [18], and overexpression of efflux transporter (P-glycoprotein) [19e21]. This led to the development of second generation (nilotinib and dasatinib) and third generation (ponatinib and bosutinib) TKIs. However, these TKIs failed to circumvent the BCR-ABL dependent drug resistance. Moreover, one important mechanism of imatinib resistance is the point mutations in the BCR-ABL kinase domain, with T315I being the most resilient to TKIs. T315I mutation occurs when threonine at amino acid position 315 (in the ABL sequence) is replaced with isoleucine, which is responsible for 15% of relapse cases of CML [22]. Unlike T315I, most of the other point mutations can be completely eradicated by the rational combination of second and third generation TKIs. Thus, there is an indispensable need to develop novel BCR-ABL T315I mutant inhibitors. We investigated whether or not PBA2 possesses anti-cancer activity against a wide range of BCR-ABL mutants including the gatekeeper residue, (the T315I mutant). We looked at the cellular effects of PBA2 such as, cell cycle arrest, induction of apoptosis and production of reactive oxygen species (ROS) in both wild type (WT) and mutant BaF3 cells. Materials and methods Chemicals and equipments PBA2 (9-(2-chloro-phenyl)-6-ethyl-1-methyl-2,4-dihydro-2,3,4,7,10-pentaazabenzo[f]azulene) was synthesized and its purity is more than 99%. The molecular weight of PBA2 is 337.806. A stock solution of PBA2 in DMSO was prepared, from which a series of dilutions were made. Imatinib mesylate was purchased from TSZ Chem. (Lexington, MA). Fig. 1A and B shows the chemical structure of PBA2 and imatinib, respectively. Roswell Park Memorial Institute Media 1640 (RPMI 1640), fetal bovine serum (FBS), phosphate buffer saline (PBS), 10,000 IU/ml penicillin and 10,000 mg/ml streptomycin were purchased from Hyclone(Waltham, Ma). Monoclonal antibody against b-actin and antibodies against BCR-ABL and p-BCR-ABL, pSTAT5, and p-Crkl were purchased from Cell Signaling Technology Inc (Beverly, MA). Superscript II reverse transcriptase enzyme, random and oligo primers, and SYBR select master mix were purchased from Invitrogen life technologies (Grand Island, NY). The primers were purchased form fisher scientific. Propidium Iodide (PI)/RNase staining buffer was purchased from BD biosciences (San Jose, CA) and the Apoptosis kit was purchased from Biotium (Hayward, CA). 5-(and-6)-chloromethyl-20 ,7'dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) was purchased from Molecular probes™(Oregon, USA). 3-(4, 5-dimethylthiazol-yl)-2,5diphenyllapatinibrazolium bromide) (MTT), Dimethyl sulfoxide (DMSO), and other chemicals were obtained from Sigma-Aldrich Chemical Co. USA. Opsys microplate reader was purchased from DYNEX TECHNOLOGIES, USA, the BD Accuri™ C6 flow cytometer was purchased from BD Biosciences (San Jose, CA), and the Aria Mx RealTime PCR System was purchased from Agilent technologies (Santa Clara, CA). Cell lines and cell culture The BaF3/WT, BaF3/Parental, BaF3/Empty vector and the mutant types BaF3/ T315I, BaF3/G250E, BaF3/M244V, BaF3/M351T, BaF3/Q252H, BaF3/E255K, BaF3/ H296P, BaF3/Y253F, BaF3/H396R, BaF3/F359V, BaF3/F311L, BaF3/E255V, and BaF3/

Fig. 1. Chemical structure of PBA2 (9-(2-chloro-phenyl)-6-ethyl-1-methyl-2,4dihydro-2,3,4,7,10-pentaaza-benzo[f]azulene) (A) and imatinib 4-[(4-methyl-1piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]phenyl]benzamide (B).

F317L were used in this study. Cells were derived by retroviral transfection using the mammalian expression vector as described previously [23,24]. All cell lines were cultured at 37  C, 5% CO2 with RPMI 1640, supplemented with 10% FBS and 1% penicillin/streptomycin. Table 1 lists all the cell lines along with the description of amino acid substitution. Cell proliferation assay The anti-proliferative effects of PBA2 were determined by a modified 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) colorimetric assay. Cells were seeded onto 96-well plates at a density of 4  103 cells per well. After 24 h of incubation, the cells were treated with either PBA2 or imatinib at the indicated concentrations (0.1e100 mM). After 72 h, 20 ml MTT (4 mg/ml) was added to each well and the plates were further incubated at 37  C for 4 h. Subsequently, the plates were centrifuged at 3000 rpm for 3 min to ensure cell adherence and formation of a monolayer of drug treated cells. The MTT with medium was removed from each well and 100 ml of DMSO was added for dissolving the formazan crystals formed by the reduction of MTT in mitochondria of proliferating cells. The absorbance was measured at 570 nm by an Opsys microplate reader (Dynex Technologies, USA). Preparation of total cell lysates The cells were incubated with various concentrations of PBA2 and imatinib for 72 h. Subsequently, cells were harvested and suspended in PBS, centrifuged at 2000 rpm for 3 min, followed by two washings with PBS. The lysis buffer (1X PBS, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 100 mg/ml p-aminophenylmethylsulfonyl fluoride) and 1% aprotinin was added to the suspension followed by vortexing for few seconds. The resuspended cells were kept on ice for 30 min, followed by centrifugation at 12,000 rpm for 20 min. The supernatant was separated and stored in 80  C for the experiment. Protein concentrations in the lysates were determined by the bicinchonic acid based protein assay [25]. Western blot analysis Equal amounts of total cell lysates (40 mg protein) were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated in a blocking solution (5% skim milk) in TBST buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature. Subsequently, the membranes were immunoblotted overnight with primary monoclonal antibodies against either b-actin at 1:1000 dilution or BCR-ABL, p-BCR-ABL, p-STAT5, and p-Crkl at 1:500 dilution, at 4  C. The membranes were then incubated for 2 h at room temperature with HRP-conjugated secondary antibody (1:1000 dilution). The proteinantibody complex was detected using chemiluminescence (Amersham, NJ). The bactin was used to confirm equal loading in each lane. The protein expression was quantified by ImageJ 1.47v Software (NIH, USA). Real-time PCR Total RNA was extracted using TRIzol® Reagent (Invitrogen life technologies) according to the manufacturer's instructions. cDNA was transcribed from RNA using Superscript II, reverse transcription kit. RT-PCR was performed using an Aria Mx Real-Time PCR System (Agilent technologies) and quantification was done using the SYBR select master mix with 2.5 ml cDNA in a final reaction volume of 10 ml. Melting curve analysis was performed to validate the primers and eliminate any possibility of primer-dimer formation. The thermal profile followed was rapid heating to 95  C to ensure DNA denaturation, followed by cooling to 60  C. Relative quantification of

Table 1 Representation of the mutant cell lines used. Name of the cell line

Description of substitution

BaF3/T315I BaF3/G250E BaF3/M244V BaF3/M351T BaF3/Q252H BaF3/E255K BaF3/H296P BaF3/Y253F BaF3/H396R BaF3/F359V BaF3/F311L BaF3/E255V BaF3/F317L

Threonine at 315 substituted by Isoleucine Glycine at 250 substituted by Glutamic acid Methionine at 244 substituted by Valine Methionine at 351 substituted by Tyrosine Glutamine at 252 substituted by Histidine Glutamic acid at 255 substituted by Lysine Histidine at 296 substituted by Proline Tyrosine at 253 substituted by Phenylalanine Histidine at 396 substituted by Arginine Phenylalanine at 359 substituted by Valine Phenylalanine at 311 substituted by Leucine Glutamic acid at 255 substituted by Valine Phenylalanine at 317 substituted by Leucine

A total of thirteen mutant BaF3 cell lines were used. Each cell line expressed a mutant form of BCR-ABL. The table represents all the cell lines along with a description of the substitution of amino acids.

Please cite this article in press as: P. Gupta, et al., PBA2, a novel inhibitor of imatinib-resistant BCR-ABL T315I mutation in chronic myeloid leukemia, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.09.025

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

CAN13048_proof ■ 7 October 2016 ■ 3/10

P. Gupta et al. / Cancer Letters xxx (2016) 1e10 p-BCR-ABL was performed using the 2DDCt method [26]. Supplemental Fig. 2 shows the sequence of primers used in this study. Cell cycle analysis The cells were incubated with PBA2 at 3 mM for different time periods (0, 24, 48, and 72 h). The cells were collected at the end of each time period and were washed twice with PBS supplemented with 0.5% bovine serum albumin (BSA) at 4  C. The cell pellet was fixed overnight in ice cold 70% ethanol at 4  C. The fixed cells were centrifuged at 500 rpm for 3 min and the suprernatant was removed. After washing with PBS supplemented with 0.5% bovine serum albumin (BSA) at 4  C, the cells were subsequently stained with 50 mg/mL PI and 100 mg/ml RNase for 1 h at room temperature in the dark. The flow cytometric analysis was performed to determine the percentage of cells in specific phases of the cell cycle(G0/G1, S, and G2/M) at FL-2 of a BD Accuri™ C6 flow cytometer (San Jose, CA). Apoptosis analysis The cells were incubated with PBA2 at 3 mM for 0, 24, 48, and 72 h. The cells were harvested and washed, and incubated with FITC-labeled annexin-V (AV, BD Pharmingen, San Diego, CA, USA) and PI, at 37  C for 30 min. Flow cytometric analysis was performed at FL-1 and FL-2 of the BD Accuri™ C6 flow cytometer to determine the apoptotic cell population. Intracellular ROS measurement The measurement of intracellular ROS levels was done by using CM-H2DCFDA (Molecular Probes). CM-H2DCFDA enters into the cells and gets converted into the fluorescent (5-chloromethyl-20 -7’-dichlorofluorescein (DCF)) product. This conversion is mediated by the action of intracellular peroxides. BaF3/WT and BaF3/T315I cells were incubated with or without PBA2 for the indicated time and collected by centrifugation. Cells were subsequently washed with 1X PBS, resuspended in 1X PBS containing 10 mM CM-H2DCFDA and incubated for 30 min in the dark at 37  C. The stained cells were washed with 1X PBS and read on the FL-1 channel of the BD Accuri™ C6 flow cytometer (San Jose, CA). Statistical analysis All experiments were repeated at least three times and the differences were determined using the two-tailed student's t-test (Microsoft Excel 2010) and statistical significance was determined at p < 0.05.

Results PBA2 inhibits cellular proliferation of a wide array of BaF3 cells In order to understand the effect of PBA2 on cell viability, a modified MTT assay was carried out. PBA2 and imatinib were screened against a panel of sixteen cell lines (as mentioned in ‘cell lines and cell culture’). As shown in Fig. 2B, PBA2 significantly reduced the cell viability of BaF3/T315I cells as compared to imatinib. PBA2 was 7-fold more potent than imatinib (IC50 PBA2 ¼ 3 mM

3

vs IC50 imatinib ¼ 21 mM) (Table 2). These results suggest that PBA2 is effective against the imatinib-resistant T3151 mutant. PBA2 exerted similar but less potent effects on various other mutant cell lines including BaF3/G250E (Fig. 3A), BaF3/M244V (Fig. 3B), BaF3/M351T (Fig. 3C), BaF3/Q252H (Fig. 3D), BaF3/E255K (Fig. 3E), and BaF3/H296P (Fig. 3F). These findings indicate that PBA2 has a wide spectrum of activity on BCR-ABL mutants as compared to imatinib. Interestingly, PBA2 did not have any significant effect on the cell viability of BaF3/WT (Fig. 2A), BaF3/Parental and BaF3/Empty vector (Supplemental Fig. 1A and B) cells, as compared to imatinib suggesting that PBA2 has a mutant specific action. Moreover, PBA2 was less potent to BaF3/Y253F, BaF3/ H396R, BaF3/F359V, BaF3/F311L, BaF3/E255V, and BaF3/F317L cells, compared to imatinib (Supplemental Fig. 1). Table 2 represents the IC50 values of PBA2 and imatinib as obtained from the MTT assay for all the sixteen cell lines. Effect of PBA2 on the BCR-ABL signaling pathways in BaF3/WT and BaF3/T315I cells To determine whether the anti-proliferative effects of PBA2 were dependent on inhibition of BCR-ABL activity, protein Table 2 IC50 values for PBA2 and imatinib on BaF3 cell lines. BaF3 cell lines

PBA2 IC50 (mM) ± SD

BaF3/WT BaF3/T315I BaF3/parental BaF3/empty vector BaF3/G250E BaF3/M244V BaF3/M351T BaF3/Q252H BaF3/E255K BaF3/H296P BaF3/Y253F BaF3/H396R BaF3/F359V BaF3/F311L BaF3/E255V BaF3/F317L

3.00 3.06 6.63 8.38 2.94 0.73 2.18 0.23 0.55 2.36 9.65 7.17 0.79 2.87 6.55 0.80

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.4 1.1 0.9 0.6 0.6 0.05 0.3 0.09 0.04 0.03 0.7 0.5 0.2 0.2 1.2 0.1

Imatinib IC50 (mM) ± SD 0.64 ± 0.04 21.01 ± 0.7 10.45 ± 1.5 12.04 ± 1.3 6.57 ± 0.7 1.80 ± 0.1 2.56 ± 0.2 0.42 ± 0.1 0.72 ± 0.07 2.40 ± 0.2 9.41 ± 0.4 0.93 ± 0.03 0.38 ± 0.4 0.233 ± 0.04 0.845 ± 0.06 0.08 ± 0.09

IC50 values obtained from cell proliferation assays.IC50 (mM) ± SD: The drug concentration that inhibited cell survival by 50% (means ± SD). Values in the table are representative of at least 3 independent experiments, each performed in triplicate.

Fig. 2. Effect of PBA2 on viability of BaF3/WT and BaF3/T315I cells. Comparative viability of BaF3/WT (A) and BaF3/T315I (B) cells treated with PBA2 (blue) and Imatinib (red). Cells were seeded in 96-well culture plates, and were incubated with various concentrations of PBA2 and Imatinib. Cell viability was determined by the MTT assay as described in “Materials and Methods”. Points with error bars represent the mean ± SD. The above figures are representative of three independent experiments, each done in triplicates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: P. Gupta, et al., PBA2, a novel inhibitor of imatinib-resistant BCR-ABL T315I mutation in chronic myeloid leukemia, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.09.025

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 4 P. Gupta et al. / Cancer Letters xxx (2016) 1e10

CAN13048_proof ■ 7 October 2016 ■ 4/10

Please cite this article in press as: P. Gupta, et al., PBA2, a novel inhibitor of imatinib-resistant BCR-ABL T315I mutation in chronic myeloid leukemia, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.09.025

Fig. 3. Effect of PBA2 on viability of mutant BaF3 cells. Comparative viability of BaF3/G250E (A), BaF3/M244V (B), BaF3. M351T (C), BaF3/Q252H (D), BaF3/E255K (E), BaF3/H296P (F) cells treated with PBA2 (blue) and Imatinib (red). Cells were seeded in 96-well culture plates, and were incubated with various concentrations of PBA2 and Imatinib. Cell viability was determined by the MTT assay as described in “Materials and Methods”. Points with error bars represent the mean ± SD. The above figures are representative of three independent experiments, each done in triplicates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

CAN13048_proof ■ 7 October 2016 ■ 5/10

P. Gupta et al. / Cancer Letters xxx (2016) 1e10

expression levels of BCR-ABL, phosphorylated BCR-ABL (p-BCRABL) and its downstream proteins, phosphorylated STAT5 and Crkl were measured by Western blot analysis. PBA2 at 3 mM inhibited the phosphorylation of BCR-ABL (Tyr177) in both BaF3/WT and BaF3/T315I cells (Fig. 4). Likewise, phosphorylation levels of STAT5 (Tyr694) and Crkl (Tyr207) were suppressed in a dose-dependent manner (Fig. 4AeD). Contrastingly, imatinib did not affect the phosphorylation levels of BCR-ABL (Tyr177), STAT5 (Tyr694) and Crkl (Tyr207) in BaF3/T315I cells (Fig. 4B and D).

5

Effect of PBA2 on the mRNA levels of p-BCR-ABL in BaF3/WT and BaF3/T315I cells To determine the effects of PBA2 on the mRNA levels of p-BCRABL, a real-time PCR was carried out. BaF3/WT and BaF3/T315I cells were treated with PBA2 at the indicated concentrations (0.5, 1.5, and 3 mM) for 72 h. The RNA was extracted and reverse transcribed to cDNA and a real time PCR was done. As shown in Fig. 5, PBA2 at 3 mM significantly decreased the mRNA expression of p-BCR-ABL in both BaF3/WT and BaF3/T315I cells. These results were consistent

Fig. 4. Effect of PBA2 on the BCR-ABL signaling pathway. Cells were treated with PBA2 (0.5e3 mM) or Imatinib (3 mM) for 72 h. Western blot experiments for p-BCR-ABL, BCR-ABL, pCrkl, and p-STAT5 were performed on BaF3/WT (A) and BaF3/T315I (B) cell lysates. Protein expression was quantified by ImageJ 1.47v Software (C and D). *p < 0.05 versus control group.

Please cite this article in press as: P. Gupta, et al., PBA2, a novel inhibitor of imatinib-resistant BCR-ABL T315I mutation in chronic myeloid leukemia, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.09.025

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

CAN13048_proof ■ 7 October 2016 ■ 6/10

6

P. Gupta et al. / Cancer Letters xxx (2016) 1e10

Fig. 5. Effect of PBA2 on the mRNA expression of p-BCR-ABL. Total RNA was extracted using TRIzol® Reagent according to the manufacturer's instructions. cDNA was transcribed and RT-PCR was performed as mentioned in “Materials and Methods”. (A) Relative p-BCR-ABL mRNA expression in BaF3/WT cells, (B) Relative p-BCR-ABL mRNA expression in BaF3/ T315I cells, treated with PBA2 (0.5e3 mM) or Imatinib (3 mM) for 72 h. Change in gene expression is denoted as relative change (ratio of target gene/reference gene). 18S was used as reference gene. Each data point represents the mean ± SD of three independent experiments. *p < 0.05 versus control group.

to the effects on the protein level of p-BCR-ABL as observed by the Western blot analysis. PBA2 induces sub G1 phase in BaF3/WT and BaF3/T315I cells In order to better understand the anti-cancer mechanism of PBA2, its effect on BaF3/WT and BaF3/T315I cell cycle progression were determined. PBA2 at 3 mM had similar effects on both BaF3/ WT and BaF3/T315Icells. In BaF3/WT cells, a sub G1 peak appeared and there was a constant increase in the percentage of cells in this phase upto 72 h (Fig. 6A). Similarly, the BaF3/T315I cells had a sub G1 peak that showed a constant increase in cell percentage upto 72 h (Fig. 6B). These findings suggest that PBA2 induces the sub G1 phase (early apoptosis) with no major effect on the cell cycle phases (Supplemental Fig. 3A and B). PBA2 induces apoptotic cell death in BaF3/WT and BaF3/T315I cells To examine whether the inhibitory effects of PBA2 are associated with induction of apoptosis, BaF3/WT and BaF3/T315I cells were treated upto 72 h. The majority of the BaF3/WT and BaF3/ T315I cells were viable in the control group and showed no or minimal signs of apoptosis (Fig. 6C and D). After incubation with PBA2 (3 mM), there was a significant increase in apoptosis in both BaF3/WT and BaF3/T315I cells. In BaF3/WT cells, there was a pronounced increase in apoptosis at 48 h and 72 h (Fig. 6C) time periods, as evident from increase in AVþ/PI population. However, in BaF3/T315I cells, the increase in AVþ/PI population was significant after 72 h (Fig. 6D) time point. PBA2 elevates ROS production on BaF3/WT and BaF3/T315I cells To investigate whether PBA2-induced apoptosis and subsequent cytotoxicity involves production of ROS, ROS levels were measured using flow cytometer. As shown in Fig. 7, PBA2 at 3 mM elevated ROS production in both BaF3/WT and BaF3/T315I cells. There was a significant increase in the ROS production from 24 h (3.13%) to 48 h (36.5%) in BaF3/WT cells, which further increased after 72 h (58.3%) (Fig. 7A and B and Supplemental Fig. 4A). Likewise, in BaF3/T315I

cells, a significant increase in ROS production was observed after 24 h (13.7%) of PBA2 treatment (3 mM), which subsequently increased on 48 h(54.6%) and 72 h (65.5%) (Fig. 7C and D and Supplemental Fig. 4B). Discussion The occurrence and progression of CML has been correlated to the presence of Phþ chromosome [27]. The ability of the BCR-ABL oncogene to undergo transformation and develop mutant forms, that exhibit the BCR-ABL phenotype but at the same time shows resistant characters, has been a major factor to combat CML [28]. Over the years, a number of strategies have been developed to fight against the mutational forms of BCR-ABL oncogene, out of which, tyrosine kinase therapy had advanced successfully in the clinics [29,30]. Since the approval of imatinib in 2001, a number of TKIs have been approved for treatment of CML. Despite of the increased survival of patients, a number of patients remain unresponsive to the TKI therapy (particularly to imatinib), thus, giving rise to the mutational forms and the problem of TKI resistance [31]. Despite the clinical efficacy of the first, second and third generation BCReABL inhibitors, many major problems persist; long-term tolerability, side effect induced by the inhibitors, resistance to the inhibitors, inability to eradicate CML stem cells and minimal residual disease [32]. Fortunately, resistant mechanisms are illustrated in several ways and novel agents with theoretically good tolerability targeting multiple signaling pathways have been designed to sensitize cancer cells to BCReABL inhibitors [1,9,32]. Although most of these inhibitors have significant ability to inhibit or reverse BCReABL resistance, long-term tolerability and efficacy remains to be tested in clinics. Recently, two benzothiazolderivates have been identified as novel BCR-ABL inhibitors [33,34]. These inhibitors showed a class effect against the cellular proliferation of the T315I mutation, as compared to the first generation TKI, imatinib. Furthermore, these agents inhibited the BCR-ABL signaling pathway, induced cell cycle arrest and caused apoptosis of BaF3/WT and BaF3/T315I cells [33,34].

Please cite this article in press as: P. Gupta, et al., PBA2, a novel inhibitor of imatinib-resistant BCR-ABL T315I mutation in chronic myeloid leukemia, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.09.025

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

CAN13048_proof ■ 7 October 2016 ■ 7/10

P. Gupta et al. / Cancer Letters xxx (2016) 1e10

7

Fig. 6. Effect of PBA2 on the cell cycle and apoptosis of BaF3/WT and BaF3/T315I cells. BaF3/WT and BaF3/T315I cells were treated with PBA2 (3 mM) in a time-dependent manner, stained with propidium iodide (PI) or annexin-V, and analyzed by flow cytometer for cell cycle and apoptosis. Quantification of cell population in sub G1 phase of BaF3/WT (A) and BaF3/T315I cells (B) Induction of apoptosis in BaF3/WT (C) and BaF3/T315I cells (D)*p < 0.05 versus control group.

PBA2 was developed to target BCR-ABL mutations and to be specifically active against the T315I mutant. In the absence of the T315I mutation, imatinibis known to form a hydrogen bond with threonine at amino acid 315 [35]. The substitution of isoleucine prevents imatinib from forming the hydrogen bond within the kinase domiain, and thereby produces resistance [18]. Furthermore, isoleucine produces steric hindrance and adds further to the resistance to imatinib [36]. PBA2 is able to overcome isoleucine induced steric hindrance and is thus effective against T315I mutation (Fig. 1A and B). To our knowledge, this is the first study to report the effects of PBA2 on a panel of mutant BaF3 cells and highlighting its potential to be an effective drug candidate against CML. Cell proliferation assay was conducted with three specific aims; 1) to know the effect of PBA2 on the cell viability of T315I mutant cells, as compared with imatinib, 2) to determine whether PBA2's effect is mutation specific or not (testing it against the parental, empty vector, and wild type cells), and 3) to see if PBA2 could inhibit other BCR-ABL expressing mutants. PBA2 (3 mM) significantly inhibited cellular proliferation of BaF3/T315I cells as compared with imatinib. These results were in accordance to earlier studies, where, researchers have reported a high IC50 value of imatinib for the T315I mutant, as we got in our study [33,34,37,38]. PBA2 at aforementioned concentration was less efficacious against the cellular viability of BaF3/Parental, BaF3/Empty vector, and BaF3/ WT cells, as compared to imatinib indicating that it is specific in action against the mutant BaF3 cells expressing the BCR-ABL oncogene. Apart from the BaF3/T315I cells, PBA2 (3 mM) was effective against six other mutant cell linesBaF3/G250E, BaF3/ M244V, BaF3/M351T, BaF3/Q252H, BaF3/E255K, and BaF3/H296P. These results reflect the wide spectrum of inhibitory activity exhibited by PBA2.

Since BCR-ABL activates multiple downstream signaling molecules which promote proliferative activity of the BCR-ABL mutants, immunoblotting analysis was conducted to examine the effects of PBA2 on phosphorylation of BCR-ABL, STAT5, and Crkl. PBA2 (3 mM) significantly inhibited the protein expression levels of p-BCR-ABL, p-STAT5, and p-Crkl in BaF3/WT and BaF3/T315I cells. This indicates a decrease in Abl kinase activity, as well as a pronounced inhibition of BCR-ABL downstream targets STAT5 and Crkl in BaF3/T315I cells. As expected, imatinib inhibited the phosphorylation of BCR-ABL, STAT5, and Crkl in BaF3/WT, but not in BaF3/T315I cells. The effect on the protein expression level was then traced back to the mRNA and a real-time PCR was conducted. In consistent with the effect on the phosphorylation of BCR-ABL, PBA2 at 3 mM decreased the mRNA level of p-BCR-ABL in both BaF3/WT and BaF3/T315I cells. Inhibition of cell growth and proliferation have been associated with the modulation of cell cycle [39]. Moreover, any alteration in the normal cell cycle gives rise to tumor development and proliferation. It has been reported that anti-cancer agents can alter the cell cycle, resulting in an arrest of cells in several phases of cell cycle; causing a reduction in growth and proliferation of cancer cells [40]. Therefore, we carried out a cell cycle analysis of PBA2. Our cell cycle results indicated that PBA2 (3 mM) did not have any significant effect on the major cell cycle phases such as G1, S, G2, and M; instead it caused sub G1 cell cycle arrest. PBA2 was found to increase the sub G1 cell population upto 72 h, in both BaF3/WT and BaF3/T315I cells. Since, the appearance of sub G1/pre-apoptotic cell population indicates the induction of early apoptosis; we carried out an apoptosis analysis using BaF3/WT and BaF3/T315I cells. In both BaF3/WT and BaF3/T315I cells, substantial amount of apoptotic cell population was obtained in the quadrant 3 (Q3) of the apoptosis representing quadrants. Q3 represents a phase of

Please cite this article in press as: P. Gupta, et al., PBA2, a novel inhibitor of imatinib-resistant BCR-ABL T315I mutation in chronic myeloid leukemia, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.09.025

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

8

P. Gupta et al. / Cancer Letters xxx (2016) 1e10 Fig. 7. Effect of PBA2 on ROS production in BaF3/WT and BaF3/T315I cells. BaF3/WT (A) and BaF3/T315I (C) cells were treated with PBA2 (3 mM) in a time-dependent manner as mentioned in “Materials and Methods”. Quantification of DCF (5-chloromethyl-20 -7’-dichlorofluorescein) positive cells (B and D).

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

CAN13048_proof ■ 7 October 2016 ■ 8/10

Please cite this article in press as: P. Gupta, et al., PBA2, a novel inhibitor of imatinib-resistant BCR-ABL T315I mutation in chronic myeloid leukemia, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.09.025

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

CAN13048_proof ■ 7 October 2016 ■ 9/10

P. Gupta et al. / Cancer Letters xxx (2016) 1e10

increase in AVþ/PI population, thus, giving an early apoptotic cell population [41]. Our results showed a constant increase in early apoptosis of both BaF3/WT and BaF3/T315I cells, thus, supporting the early findings from the cell cycle analysis. Although induction of apoptosis and cancer cell growth are two opposing phenomena, but generation of ROS have known to be implicated in both [42]. Generation of intracellular ROS in cancer cells causes an alteration in the cellular macromolecules promoting apoptosis [42,43]. Therefore, we identified whether PBA2 promotes ROS in BaF3/WT and BaF3/T315I cells. Our results showed that PBA2 at 3 mM significantly increased ROS in both BaF3/WT and BaF3/T315I cells. In conclusion, PBA2, a novel BCR-ABL TKI, surmounts imatinib resistance in BaF3/T315I cells. Our results showed that PBA2 is effective in producing cellular toxicity and altering the normal cell cycle events of BaF3/WT and BaF3/T315I cells, which may be of great interest in clinics. Given its cellular mechanism of action and inhibition of a wide array of BCR-ABL mutants, PBA2 could be developed as a potential agent to overcome imatinib resistance in CML. Funding This work was supported by the St. John's University Research Seed Grant (No. 579-1110-7002) for Zhe-Sheng Chen and Guangdong scientific and technological international collaborative foundation (No. 2013B051000046) and Guangzhou scientific technology and innovation foundation (No. 201601010008 and No. 201504010038) for Li-Wu Fu. Conflict of interest None declared. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.canlet.2016.09.025. References [1] X. An, A.K. Tiwari, Y. Sun, P.R. Ding, C.R. Ashby Jr., Z.S. Chen, BCR-ABL tyrosine kinase inhibitors in the treatment of Philadelphia chromosome positive chronic myeloid leukemia: a review, Leuk. Res. 34 (2010) 1255e1268. [2] Y. Chen, C. Peng, D. Li, S. Li, Molecular and cellular bases of chronic myeloid leukemia, Protein Cell 1 (2010) 124e132. [3] S. Faderl, H.M. Kantarjian, M. Talpaz, Chronic myelogenous leukemia: update on biology and treatment, Oncol. (Williston Park) 13 (1999) 169e180 discussion 181, 184. [4] H.M. Kantarjian, A. Deisseroth, R. Kurzrock, Z. Estrov, M. Talpaz, Chronic myelogenous leukemia: a concise update, Blood 82 (1993) 691e703. [5] C.E. DeSantis, C.C. Lin, A.B. Mariotto, R.L. Siegel, K.D. Stein, J.L. Kramer, R. Alteri, A.S. Robbins, A. Jemal, Cancer treatment and survivorship statistics, 2014, CA Cancer J. Clin. 64 (2014) 252e271. [6] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2016, CA Cancer J. Clin. 66 (2016) 7e30. [7] X. Huang, J. Cortes, H. Kantarjian, Estimations of the increasing prevalence and plateau prevalence of chronic myeloid leukemia in the era of tyrosine kinase inhibitor therapy, Cancer 118 (2012) 3123e3127. [8] J. Erikson, C.A. Griffin, A. ar-Rushdi, M. Valtieri, J. Hoxie, J. Finan, B.S. Emanuel, G. Rovera, P.C. Nowell, C.M. Croce, Heterogeneity of chromosome 22 breakpoint in Philadelphia-positive (Phþ) acute lymphocytic leukemia, Proc. Natl. Acad. Sci. U. S. A. 83 (1986) 1807e1811. [9] E. Weisberg, P.W. Manley, S.W. Cowan-Jacob, A. Hochhaus, J.D. Griffin, Second generation inhibitors of BCR-ABL for the treatment of imatinib-resistant chronic myeloid leukaemia, Nat. Rev. Cancer 7 (2007) 345e356. [10] C.R. Bartram, A. de Klein, A. Hagemeijer, T. van Agthoven, A. Geurts van Kessel, D. Bootsma, G. Grosveld, M.A. Ferguson-Smith, T. Davies, M. Stone, et al., Translocation of c-ab1 oncogene correlates with the presence of a Philadelphia chromosome in chronic myelocytic leukaemia, Nature 306 (1983) 277e280. [11] A.S. Advani, A.M. Pendergast, Bcr-Abl variants: biological and clinical aspects, Leuk. Res. 26 (2002) 713e720.

9

[12] L.C. Chan, K.K. Karhi, S.I. Rayter, N. Heisterkamp, S. Eridani, R. Powles, S.D. Lawler, J. Groffen, J.G. Foulkes, M.F. Greaves, L.M. Wiedemann, A novel abl protein expressed in Philadelphia chromosome positive acute lymphoblastic leukaemia, Nature 325 (1987) 635e637. s-Cardama, H. Kantarjian, J. Cortes, Flying under the radar: the new [13] A. Quinta wave of BCR-ABL inhibitors, Nat. Rev. Drug Discov. 6 (2007) 834e848. [14] C.L. Sawyers, A. Hochhaus, E. Feldman, J.M. Goldman, C.B. Miller, O.G. Ottmann, C.A. Schiffer, M. Talpaz, F. Guilhot, M.W. Deininger, T. Fischer, S.G. O'Brien, R.M. Stone, C.B. Gambacorti-Passerini, N.H. Russell, J.J. Reiffers, T.C. Shea, B. Chapuis, S. Coutre, S. Tura, E. Morra, R.A. Larson, A. Saven, C. Peschel, A. Gratwohl, F. Mandelli, M. Ben-Am, I. Gathmann, R. Capdeville, R.L. Paquette, B.J. Druker, Imatinib induces hematologic and cytogenetic responses in patients with chronic myelogenous leukemia in myeloid blast crisis: results of a phase II study, Blood 99 (2002) 3530e3539. [15] M. Talpaz, R.T. Silver, B.J. Druker, J.M. Goldman, C. Gambacorti-Passerini, F. Guilhot, C.A. Schiffer, T. Fischer, M.W. Deininger, A.L. Lennard, A. Hochhaus, O.G. Ottmann, A. Gratwohl, M. Baccarani, R. Stone, S. Tura, F.X. Mahon, S. Fernandes-Reese, I. Gathmann, R. Capdeville, H.M. Kantarjian, C.L. Sawyers, Imatinib induces durable hematologic and cytogenetic responses in patients with accelerated phase chronic myeloid leukemia: results of a phase 2 study, Blood 99 (2002) 1928e1937. [16] R.J. Kathawala, P. Gupta, C.R. Ashby, Z.S. Chen, The modulation of ABC transporter-mediated multidrug resistance in cancer: a review of the past decade, Drug Resist Updat 18 (2015) 1e17. [17] C. Barthe, P. Cony-Makhoul, J.V. Melo, J.R. Mahon, Roots of clinical resistance to STI-571 cancer therapy, Science 293 (2001) 2163. [18] M.E. Gorre, M. Mohammed, K. Ellwood, N. Hsu, R. Paquette, P.N. Rao, C.L. Sawyers, Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification, Science 293 (2001) 876e880. [19] C. Gambacorti-Passerini, M. Zucchetti, D. Russo, R. Frapolli, M. Verga, S. Bungaro, L. Tornaghi, F. Rossi, P. Pioltelli, E. Pogliani, D. Alberti, G. Corneo, M. D'Incalci, Alpha1 acid glycoprotein binds to imatinib (STI571) and substantially alters its pharmacokinetics in chronic myeloid leukemia patients, Clin. Cancer Res. 9 (2003) 625e632. [20] K.L. Silva, P.S. de Souza, G. Nestal de Moraes, A. Moellmann-Coelho, C. Vasconcelos Fda, R.C. Maia, XIAP and P-glycoprotein co-expression is related to imatinib resistance in chronic myeloid leukemia cells, Leuk. Res. 37 (2013) 1350e1358. [21] P. Gupta, K.A. Jani, D.H. Yang, M. Sadoqi, E. Squillante, Z.S. Chen, Revisiting the role of nanoparticles as modulators of drug resistance and metabolism in cancer, Expert Opin. Drug Metab. Toxicol. 12 (2016) 281e289. [22] N.P. Shah, J.M. Nicoll, B. Nagar, M.E. Gorre, R.L. Paquette, J. Kuriyan, C.L. Sawyers, Multiple BCR-ABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase and blast crisis chronic myeloid leukemia, Cancer Cell 2 (2002) 117e125. [23] P. La Rosee, A.S. Corbin, E.P. Stoffregen, M.W. Deininger, B.J. Druker, Activity of the Bcr-Abl kinase inhibitor PD180970 against clinically relevant Bcr-Abl isoforms that cause resistance to imatinib mesylate (Gleevec, STI571), Cancer Res. 62 (2002) 7149e7153. [24] T. O'Hare, D.K. Walters, E.P. Stoffregen, T. Jia, P.W. Manley, J. Mestan, S.W. Cowan-Jacob, F.Y. Lee, M.C. Heinrich, M.W. Deininger, B.J. Druker, In vitro activity of Bcr-Abl inhibitors AMN107 and BMS-354825 against clinically relevant imatinib-resistant Abl kinase domain mutants, Cancer Res. 65 (2005) 4500e4505. [25] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248e254. [26] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method, Methods 25 (2001) 402e408. [27] E. Frei 3rd, J.H. Tjio, J. Whang, P.P. Carbone, Studies of the Philadelphia chromosome in patients with chronic myelogenous leukemia, Ann. N. Y. Acad. Sci. 113 (1964) 1073e1080. [28] K. Yang, L.W. Fu, Mechanisms of resistance to BCR-ABL TKIs and the therapeutic strategies: a review, Crit. Rev. Oncol. Hematol. 93 (2015) 277e292. Q3 s-Cardama, J. Cortes, Therapeutic options against BCR-ABL1 T315I[29] A. Quinta positive chronic myelogenous leukemia, Clin. Cancer Res. 14 (2008) 4392e4399. [30] P. Ramirez, J.F. DiPersio, Therapy options in imatinib failures, Oncologist 13 (2008) 424e434. [31] J.V. Melo, D.J. Barnes, Chronic myeloid leukaemia as a model of disease evolution in human cancer, Nat. Rev. Cancer 7 (2007) 441e453. [32] K. Yang, L.W. Fu, Mechanisms of resistance to BCR-ABL TKIs and the therapeutic strategies: a review, Crit. Rev. Oncol. Hematol. (2014). Q4 [33] S.M. Yun, K.H. Jung, S.J. Kim, Z. Fang, M.K. Son, H.H. Yan, H. Lee, J. Kim, S. Shin, S. Hong, S.S. Hong, HS-438, a new inhibitor of imatinib-resistant BCR-ABL T315I mutation in chronic myeloid leukemia, Cancer Lett. 348 (2014) 50e60. [34] S.J. Kim, K.H. Jung, H.H. Yan, M.K. Son, Z. Fang, Y.L. Ryu, H. Lee, J.H. Lim, J.K. Suh, J. Kim, S. Lee, S. Hong, S.S. Hong, HS-543 induces apoptosis of Imatinib-resistant chronic myelogenous leukemia with T315I mutation, Oncotarget 6 (2015) 1507e1518. [35] T. Schindler, W. Bornmann, P. Pellicena, W.T. Miller, B. Clarkson, J. Kuriyan, Structural mechanism for STI-571 inhibition of abelson tyrosine kinase, Science 289 (2000) 1938e1942.

Please cite this article in press as: P. Gupta, et al., PBA2, a novel inhibitor of imatinib-resistant BCR-ABL T315I mutation in chronic myeloid leukemia, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.09.025

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10

CAN13048_proof ■ 7 October 2016 ■ 10/10

10

P. Gupta et al. / Cancer Letters xxx (2016) 1e10

[36] N.P. Shah, C. Tran, F.Y. Lee, P. Chen, D. Norris, C.L. Sawyers, Overriding imatinib resistance with a novel ABL kinase inhibitor, Science 305 (2004) 399e401. [37] E. Weisberg, P. Manley, J. Mestan, S. Cowan-Jacob, A. Ray, J.D. Griffin, AMN107 (nilotinib): a novel and selective inhibitor of BCR-ABL, Br. J. Cancer 94 (2006) 1765e1769. [38] J. Zhang, F.J. Adri an, W. Jahnke, S.W. Cowan-Jacob, A.G. Li, R.E. Iacob, T. Sim, J. Powers, C. Dierks, F. Sun, G.R. Guo, Q. Ding, B. Okram, Y. Choi, A. Wojciechowski, X. Deng, G. Liu, G. Fendrich, A. Strauss, N. Vajpai, S. Grzesiek, T. Tuntland, Y. Liu, B. Bursulaya, M. Azam, P.W. Manley, J.R. Engen, G.Q. Daley, M. Warmuth, N.S. Gray, Targeting Bcr-Abl by combining allosteric with ATP-binding-site inhibitors, Nature 463 (2010) 501e506. [39] J.A. Pietenpol, Z.A. Stewart, Cell cycle checkpoint signaling: cell cycle arrest versus apoptosis, Toxicology 181e182 (2002) 475e481.

[40] M. Malumbres, P. Pevarello, M. Barbacid, J.R. Bischoff, CDK inhibitors in cancer therapy: what is next? Trends Pharmacol. Sci. 29 (2008) 16e21. [41] C. Cao, B. Liu, C. Zeng, Y. Lu, S. Chen, L. Yang, B. Li, Y. Li, Y. Li, A polymethoxyflavone from Laggera pterodonta induces apoptosis in imatinib-resistant K562R cells via activation of the intrinsic apoptosis pathway, Cancer Cell Int. 14 (2014) 137. s, F.M. S nez, Role of reactive oxygen species in [42] J.M. Mate anchez-Jime apoptosis: implications for cancer therapy, Int. J. Biochem. Cell Biol. 32 (2000) 157e170. [43] M.L. Circu, T.Y. Aw, Reactive oxygen species, cellular redox systems, and apoptosis, Free Radic. Biol. Med. 48 (2010) 749e762.

Please cite this article in press as: P. Gupta, et al., PBA2, a novel inhibitor of imatinib-resistant BCR-ABL T315I mutation in chronic myeloid leukemia, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.09.025

11 12 13 14 15 16 17 18 19 20