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Published OnlineFirst February 19, 2014; DOI: 10.1158/1535-7163.MCT-13-0397

Molecular Cancer Cancer Biology and Signal Transduction Therapeutics

Ponatinib Induces Apoptosis in Imatinib-Resistant Human Mast Cells by Dephosphorylating Mutant D816V KIT and Silencing b-Catenin Signaling

Bei Jin1,2, Ke Ding3, and Jingxuan Pan1,2

Abstract Gain-of-function mutations of membrane receptor tyrosine kinase KIT, especially gatekeeper D816V point mutation in KIT, render kinase autoactivation, disease progression, and poor prognosis. D816V KIT is found in approximately 80% of the patients with systemic mastocytosis, and is resistant to the first and second generations of tyrosine kinase inhibitors (TKI). The purpose of this investigation was aimed at exploring whether ponatinib (AP24534), a novel effective TKI against T315I Bcr-Abl, was active against D816V KIT. We discovered that ponatinib abrogated the phosphorylation of KIT harboring either V560G (sensitive to imatinib) or D816V mutation (resistant to imatinib) and the downstream signaling transduction. Ponatinib inhibited the growth of D816V KIT–expressing cells in culture and nude mouse xenografted tumor. Ponatinib triggered apoptosis by inducing the release of cytochrome c and AIF, downregulation of Mcl-1. Furthermore, ponatinib abrogated the phosphorylation of b-catenin at the site Y654, suppressed the translocation of b-catenin, and inhibited the transcription and DNA binding of TCF and the expression of its targets (e.g., AXIN2, c-MYC,andCCND1). Moreover, ponatinib was highly active against xenografted D816V KIT tumors in nude mice and significantly prolonged the survival of mice with aggressive systemic mastocytosis or mast cell leukemia by impeding the expansion and infiltration of mast cells with imatinib- resistant D814Y KIT. Our findings warrant a clinical trial of ponatinib in patients with systemic mastocytosis harboring D816V KIT. Mol Cancer Ther; 13(5); 1217–30. 2014 AACR.

Introduction tocytosis (1). Currently, IFN-a (2) and cladribine (2CdA) Mastocytosis is an orphan, mostly sporadic disease, constitute the treatments of choice for the first-line ther- characterized by abnormal accumulation of mast cells apy in systemic mastocytosis, however, with inadequate and activation of mast cells in various organs, involving degree and duration of response (3). Imatinib has been bone marrow, skin, liver, and gastrointestinal tract (1). approved by the U.S. Food and Drug Administration Systemic mastocytosis and cutaneous mastocytosis con- (FDA) for adult systemic mastocytosis patients without stitute two major forms of mastocytosis. The clinical the KIT D816V mutation (4). Other TKIs such as dasatinib course in patients with systemic mastocytosis ranges from (5), midostaurin (PKC412; ref. 6), semaxinib (7), and indolent and asymptomatic to a highly aggressive disease EXEL-0862 (8) have been reported to exhibit growth course, even mast cell leukemia (MCL; ref. 1). Acquired inhibitory and apoptosis induction effects in mast cells activating mutations in KIT (e.g., D816V, D816Y, and harboring D816V KIT mutation. Although Ustun and D816F) have been identified in most patients with mas- colleagues reported that dasatinib has efficacy in systemic mastocytosis-AML patients bearing D816V KIT, a phase II study from an independent group did not support the potency of dasatinib against D816V KIT (9). Although fi 1 Authors' Af liations: Department of Pathophysiology, Zhongshan midostaurin (PKC412) has a synergistic effect with dasa- School of Medicine, Sun Yat-sen University; 2Key Laboratory of Tropical Disease Control (Sun Yat-sen University), Ministry of Education; and 3Key tinib to inhibit the growth of neoplastic mast cells (6, 10), Laboratory of Regenerative Biology and Institute of Chemical Biology, the long-term benefit of PKC412 has not yet been deter- Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Science Park, Guangzhou, China mined. Therefore, development of novel effective drugs to treat aggressive systemic mastocytosis and MCL is still Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). needed. KIT is a transmembrane receptor tyrosine kinase for Corresponding Author: Jingxuan Pan, Department of Pathophysiology, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan stem cell factor (SCF; ref. 11). On the stimulation of SCF, Road II, Guangzhou 510089, People's Republic of China. Phone and fax: KIT undergoes phosphorylation at Y568 and Y570 resi- 86-20-8733 2788; E-mail: [email protected] dues and activates various signaling pathways (12). Gain- doi: 10.1158/1535-7163.MCT-13-0397 of-function mutations in KIT also lead to KIT activation 2014 American Association for Cancer Research. and give rise to uncontrolled cell growth, cell survival,

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and apoptosis resistance (13). Imatinib inhibits activation anti-actin were from Sigma-Aldrich; and anti-mouse of wild-type KIT and some point mutant KIT (e.g., V560G) immunoglobulin G and anti-rabbit immunoglobulin G but not D816V by competitively occupying a KIT enzy- horseradish peroxidase-conjugated antibodies were from matic ATP-binding pocket. Unfortunately, the kinase Pierce Biotechnology. domain mutant D816V KIT counting for approximately 80% of all patients with systemic mastocytosis is resistant Cell culture and analysis of mutation in KIT to imatinib (14). In addition, KIT mediates tyrosine phos- Imatinib-sensitive HMC-1.1 cells harboring the V560G phorylation and nuclear localization of b-catenin, which is mutation in the juxtamembane domain of KIT and ima- an important component of the Wnt/b-catenin signaling tinib-resistant HMC-1.2 cells harboring both V560G and pathway in mast cell leukemia (15, 16). The b-catenin D816V mutations were kindly provided by Dr. Joseph accumulates in cytoplasm and transfers to the nucleus, Butterfield (Mayo Clinic, Rochester, MN) in 2006 (24, 25). where it engages TCF/LEF transcription factors for genes Both subclones were grown in Iscove’s modified Dulbec- transcription (17). co’s medium (Invitrogen) supplemented with 10% fetal Ponatinib (AP24534), a potent multitargeted kinase calf serum (FCS; Hyclone) and 1.2 mmol/L a-thioglycerol inhibitor (18), has been approved by the FDA for treating (Sigma) in a 37C humidified incubator containing 5% imatinib-resistant patients with chronic myelogenous leu- CO2. The murine P815 mastocytoma cell line harboring kemia, including those harboring the gatekeeper mutant the D814Y mutation (corresponding to human D816Y; T315I Bcr-Abl (19, 20). Besides, ponatinib also manifests ref. 26) was purchased from the American Type Culture inhibitory activity toward PDGFR, KIT, as well as FGFR Collection and cultured in DMEM supplemented with (21, 22). Although it has been reported that ponatinib is 10% FCS and 25 mmol/L HEPES buffer. All the cell lines active against BaF3 cells stably expressing Y823D KIT in were tested and authenticated by using short tandem vitro (22) and D816V KIT-positive mast neoplastic cells repeat (STR) matching analysis of cells last month. No (10), the in vivo effect remains to be examined. cross-contamination of other human cells was found in all In this report, our purpose was to validate the inhibitory three lines of cells. effect of ponatinib against the mutant KIT cells and the in The presence of mutations in KIT was analyzed rou- vivo antineoplastic efficacy in mouse models bearing cells tinely every month (the last test was performed last week, with mutant KIT. We found that ponatinib inhibited the Supplementary Fig. S1) in our laboratory with the method growth of D816V KIT-expressing cells in culture and nude described previously (27). The primers of KIT are listed in mouse xenografted tumor and significantly prolonged the Supplementary Table S1. survival of mice with aggressive systemic mastocytosis or mast cell leukemia. Preparation of cell lysates Total cell lysates were prepared in radioimmunopreci- Materials and Methods pitation assay buffer. For detection of AIF and cytochrome c Reagents and antibodies , cytosolic fraction was prepared with digitonin extrac- Ponatinib (purity > 95%, high-performance liquid chro- tion buffer (28). Extracts of subcellular cytoplasmic, mito- matography) was synthesized in our laboratory, prepared chondrial, and nuclear fractions were separated as previ- as a 20 mmol/L stocking solution in dimethyl sulfoxide ously described (23). (DMSO) and stored in aliquots at 20C. Imatinib mesy- Real-time quantitative RT-PCR late was a product of Novartis Pharmaceuticals (23). Total mRNA was extracted with TRIzol reagent (Invi- Z-DEVD-fmk (caspase-3 inhibitor) was purchased from trogen), then reverse-transcribed into first-strand comple- BD Biosciences. MG132 was from EMD Biosciences. Anti- mentary DNA (cDNA) with maxima first strand cDNA bodies and their sources were as follows: mouse mono- synthesis kit (Thermo Fisher), followed by real-time quan- clonal antibodies against KIT (CD117), PARP, Bcl-2, XIAP, titative PCR with the SYBR Premix Ex Taq kit (TaKaRa) active caspase-3, cytochrome c (clone 6H2.B4), and FITC according the protocol described previously (23).The pri- mouse IgG1k isotype control were from BD Biosciences mers are shown in Supplementary Table S1. Each PCR Pharmingen; rabbit polyclonal antibodies against apopto- reaction was in a 20 mL volume on a 96-well plate in Roche sis-inducing factor (AIF), Bax, Bcl-X , Mcl-1 (S-19), and L LightCycler480. phospho-KIT (Y568/570) were from Santa Cruz Biotech- nology; anti-Bim was from Stressgen; antibodies against Cell viability assay caspase-3, -8, -9, phospho-Erk1/2 (T202/Y204), Erk1/2, The MTS assay (CellTiter 96 Aqueous One Solution phospho-Akt (Ser473), and Akt were from Cell Signaling reagent; Promega) was used to evaluate cell viability as Technology; anti-survivin was from Novus Biologicals; described previously (23, 28). Synergism of combinational antibodies against phospho-Stat3 (Y705), anti-Stat3, phos- drugs was evaluated by the median-effect method of pho-Stat5 (Y694/Y699; clone 8-5-2), anti-Stat5, and anti- Chou and Talalay (28). active-b-catenin (anti-ABC, clone 8E7) were from Upstate Technology; cytochrome c oxidase subunit II (COX II) was Apoptosis assay by flow cytometry from Molecular Probes; anti-mouse CD45-FITC and Apoptosis was measured using Annexin V-fluores- CD117-PE were from eBioscience. Cycloheximide and cein isothiocyanate (FITC) and propidium iodide (PI;

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Sigma-Aldrich) and analyzed with a FACScalibur flow (Pierce Biotechnology) following the manufacturer’s cytometer and CellQuestPro software as described (8, 23). instructions (28). The oligonucleotides for TCF were as follows: forward: 50- TGCCGGGCTTTGATCTTTG-30; Colony formation assay reverse: 50- AGCAAAGATCAAAGCCCGG-30. Human mast cell lines were initially treated with ponatinib at 1.0 to 100 nmol/L for 24 hours, harvested and In vivo antitumor effect of ponatinib washed with PBS, and subsequently seeded in methyl- For tumor xenograft experiments, male nude nu/nu cellulose medium (Methocult H4434, STEMCELL Tech- BALB/c mice were purchased from Slac Laboratory Ani- nologies) following the manufacturer’s instructions. One mal Co (Shanghai, China). HMC-1.2 cells (200 mLof30 week later, the number of colony-forming units was million cells in serum-free medium suspension) were evaluated with microscope according to the standard implanted subcutaneously into the flanks of 4- to 6- criteria (28). week-old nude mice (30). Tumor volumes were measured every 2 days. When the tumors were palpable (50 mm3), Transfection of plasmids and small interfering RNA mice were randomized according to the average volume ON-TARGET plus SMART pool siRNA duplexes of the tumors to the placebo (n ¼ 5) or experimental against human Mcl-1#1 (cat# L-004501-00-0005) and groups (n ¼ 8), and treated every 2 days by oral gavage ON-TARGET plus NON-Targeting siRNA (mock siRNA; with placebo (100 mL of NaCl 0.9% solution which con- cat# D-001810-01) were from Dharmacon RNA Technol- tains the same amount of DMSO as in the ponatinib ogies, SignalSilence siRNA duplexes against Mcl-1#2 (cat# solution) or ponatinib at 20 mg/kg (18). The animals were 6315), b-catenin #1 (cat# 6225), b-catenin #2 (cat# 6238), euthanized after 2 weeks of treatment. The mean tumor and control siRNA were from Cell Signaling Technology. weights and mean tumor volume from the last measure- pCMV5-flag-human Mcl-1 and human pcDNA3-b-cate- ment of the 2 groups were compared by a t test. The body nin were from Addgene. Plasmids, empty vector, specific weight, feeding behavior, and motor activity of each siRNA, or mock siRNA were delivered into HMC-1.2 cells animal were monitored as indicators of general health. with the Cell Line Nucleofector Kit T (Amaxa) and pro- For the murine model with aggressive systemic mas- gram O-17 following the manufacturer’s instructions (25). tocytosis (ASM)/mast cell leukemia (MCL; refs. 25, 30), 6- The cells were then exposed to ponatinib and subjected to to 8-week-old male DBA/2 mice purchased from Slac cell death assay and Western blotting analysis. Laboratory Animal Co were randomly divided into 3 groups: normal (n ¼ 5), placebo (n ¼ 8; treated with Immunofluorescence staining DMSO-containing tissue culture medium), and experi- HMC-1.2 cells were grown with 0.3 mmol/L of ponati- mental (n ¼ 8; treated with 20 mg/kg ponatinib orogastric nib for 24 hours. Immunofluorescence staining was car- gavage once daily). ried out as previously reported (8). Mice were kept under specific pathogen-free conditions in the Sun Yat-sen University animal care facility. All Assay of intracellular active b-catenin with flow animal studies were conducted with the approval of the cytometer Sun Yat-sen University Institutional Animal Care and Use Ponatinib-treated HMC-1.2 cells were harvested, fixed Committee. in 4% formaldehyde for 10 minutes at 37C, and permea- bilized in prechilled 90% methanol for 30 minutes on ice. Immunohistochemical staining After washing and blocking, the cells were stained with Formalin-fixed xenografts of HMC-1.2 cells were anti-active-b-catenin, followed by flow cytometer analysis embedded in the paraffin and sectioned. Immunohisto- after staining with the secondary antibody Alexa Fluor chemical staining of tumor xenograft sections (4 mm) was 488-conjugated goat anti-mouse IgG. FITC-conjugated carried out with Max Vision kit (Maixin Biol) following mouse IgG1 k isotype control was stained the same way the manufacturer’s instructions (23). except the secondary antibody and evaluated the back- ground fluorescence (29). Detection of mastocytoma cells by flow cytometry The mononuclear cells were prepared from spleens of Dual luciferase reporter assay ASM/MCL mice as previously described (25, 29). The cells The TCF/LEF reporter plasmid, pTOP-flash, and its were stained with anti-CD45-FITC and anti-CD117-PE, mutant control, pFOP-flash, were purchased from EMD followed by analysis with a FACSCalibur flow cytometer. Millipore. The Renilla luciferase reporter construct, pRL- TK, was purchased from Promega. The dual luciferase Statistical analysis reporter assay was performed according to the manufac- All experiments were performed at least three times, turer’s instructions (Promega; ref. 28). and data are exhibited as mean 95% confidence intervals. Comparisons between 2 groups were analyzed by Electrophoretic mobility shift assay the t test while those between more than 2 groups by one- Electrophoretic mobility shift assay (EMSA) was per- way ANOVA with post hoc comparison by the Tukey test formed using the LightShift Chemiluminescent EMSA Kit unless otherwise stated. GraphPad Prism 5.0 (GraphPad

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Software) was used for statistical analysis. P < 0.05 was downstream signaling (Stat3/5, Akt, Erk1/2). Our results considered statistically signiﬁcant. showed that ponatinib potently inhibited the phosphorylation of KIT in both dose- and time-dependent manners Results (Fig. 1B). However, ponatinib did not alter the levels of Ponatinib abolishes the phosphorylation of KIT and total protein and mRNA in KIT as detected with Western its downstream target molecules blotting analysis and qRT-PCR, respectively (Fig. 1B and We ﬁrst analyzed the effect of ponatinib (Fig. 1A) on the C). Ponatinib decreased the level of phosphorylated KIT phosphorylation of KIT at Y568/570 residues and its in P815 cells (Fig. 1D). Correspondingly, ponatinib dose-

Figure 1. Ponatinib blocks the phosphorylation of KIT and its downstream target molecules. A, chemical structure of ponatinib. B, ponatinib inhibited the phosphorylation of KIT in a dose- or time-dependent manner in HMC cell lines. C, ponatinib did not alter the transcription of KIT. HMC cells were treated with ponatinib for 24 hours and RNAs were extracted and subjected to real-time RT-PCR analysis. D, ponatinib inhibited the phosphorylation of KIT in P815 cells. E, ponatinib inhibited the downstream signaling of KIT. Stat3, Stat5, Akt, and Erk1/2 were analyzed in the cell lysates from cells with the same treatment in Fig. 1B.

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and time-dependently inhibited the phosphorylation of between ponatinib and rapamycin based on the combi- Stat3, Stat5, Akt, and Erk1/2, which are downstream nation index (CI; Fig. 2B; ref. 28). targets of KIT (Fig. 1E). Taken together, these results To evaluate the impact of ponatinib on anchorage- suggest a strikingly inhibitory effect of ponatinib on KIT independent growth of the mast cells, we performed the signaling. colony formation assay in methylcellulose. Mast cells were exposed to increasing concentrations of ponatinib Ponatinib inhibits the growth of mast cells harboring for 24 hours and then assayed for colony formation in D816V KIT drug-free culture. Ponatinib potently inhibited the colony We next determined the effect of ponatinib on the formation in methylcellulose in a dose-dependent man- growth of the mast cells. The presence of D816V mutation ner (Fig. 2C). in KIT leads to resistance to imatinib (14). We confirmed the differential sensitivity of HMC-1.1 cells and HMC-1.2 Ponatinib induces apoptosis in mast cells harboring m > c cells to imatinib, with IC50 values of 1.1 mol/L versus 20 D816V KIT by triggering release of cytochrome and mmol/L (Fig. 2A). In contrast, ponatinib significantly apoptosis-inducing factor inhibited the growth of both imatinib-resistant HMC- We evaluated the capability of ponatinib to induce 1.2 and -sensitive HMC-1.1 cells, with IC50 values of apoptosis with flow cytometry. Ponatinib induced signif- 165 and 143 nmol/L, respectively (Fig. 2A). Furthermore, icantly increased cell death in both imatinib-sensitive ponatinib inhibited the viability of the murine P815 cells at HMC-1.1 cells and -resistant HMC-1.2 cells to a similar an IC50 of 72 nmol/L (Fig. 2A). degree ( 55% vs. 65%; Fig. 3A). Because simultaneously inhibiting a specific tyrosine Western blotting analysis showed that ponatinib dose- kinase and its downstream targets such as mTOR usually and time-dependently induced specific cleavage of PARP, results in synergism (31, 32), we examined the effect of the caspase-8, -9, and -3 in the malignant mast cells (Fig. 3B), combination between ponatinib and rapamycin, an further confirming the occurrence of apoptosis. Of note, mTOR inhibitor, in human mast cells. The results assessed when detecting cytochrome c and AIF in the cytosolic by MTS assay after 72 hours of incubation in a serially fraction, their levels were increased as early as 12 hours diluted mixture at a fixed ratio revealed synergism after 0.3 mmol/L ponatinib treatment (Fig. 3C), which

Figure 2. Ponatinib inhibits the growth of human mast cells harboring D816V KIT. A, HMC-1.1, HMC-1.2, and P815 cells were incubated in escalating concentrations of ponatinib or imatinib for 72 hours followed by MTS assay. Dose–response curves with mean 95% confidence intervals are shown. B, synergistic effect of the combination of ponatinib and rapamycin in mast cells was observed by MTS assay after incubating cells with a serial diluted mixture at a fixed ratio of the two drugs. CI is plotted against the fraction affect. The reference line indicates CI ¼ 1. CI values below 1 indicate synergism between the two drugs. C, clonogenicity of human mast cells in methylcellulose was inhibited in a dose-dependent manner. Curves are plotted with mean 95% confidence intervals.

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Figure 4. Mcl-1 plays an important part in the apoptosis induced by ponatinib. A, the levels of Mcl-1 significantly affected the cell fate in ponatinib- treated cells. HMC-1.2 cells were transfected with either Mcl-1 plasmid or siRNA and then treated with or without ponatinib. Cell death assay (Trypan blue exclusion, bottom) and immunoblotting analysis of PARP cleavage were performed. , P < 0.05; , P < 0.01; , P < 0.0001. B, ponatinib accelerated the turnover rate of Mcl-1 protein. Data shown are one representative Western blotting analysis of Mcl-1 protein and curves of integrated density 100% normalized to the control band, which were analyzed by ImageJ from three independent experiments. Data, mean 95% confidence intervals. , P < 0.001, comparisons were made by the t test between points A and A0 and B and B0. C, MG132 did not reverse Mcl-1 degradation caused by ponatinib treatment. Western blotting analysis of human mast cells incubated with 0.3 mmol/L in the presence or absence of 1.0 mmol/L MG132 is shown. D, the conversion of p42 Mcl-1 to p28 Mcl-1 induced by ponatinib was not abrogated by Z-DEVD-fmk. obviously preceded the activation of caspase-9 and Effect of ponatinib on the expression of apoptosis- caspase-3. These results suggested that ponatinib might related proteins lead to damage of mitochondria and trigger the intrinsic To figure out the mechanism of ponatinib-induced pathway of apoptosis. apoptosis, we examined the expression of apoptosis-

Figure 3. The effects of ponatinib on the apoptosis-related proteins. A, ponatinib induced apoptosis in human mast cells. HMC-1.1 and HMC-1.2 cells were subjected to the Annexin V-FITC-PI double staining and flow cytometry analysis after exposure to escalating concentrations for 24 hours, or to 0.3 mmol/L ponatinib for the different times indicated. Representative flow cytometry dot plots for each treatment and bar plots with mean 95% confidence intervals from 3 independent experiments are shown. The y-axisisthesumofthetopleft,topright, and bottom right quadrants. , P < 0.05; , P < 0.01; , P < 0.0001, one-way ANOVA with post hoc intergroup comparison by the Tukey test. B, dose- and time-course Western blotting analysis of PARP cleavage, the activation of caspase-3, -8, and -9 in the whole-cell lysates of human mast cells are shown. C, ponatinib induced the release of apoptosis-inducing factor (AIF) and cytochrome c from the mitochondria. COX II served as a mitochondrial marker for assessment of contamination of cytosolic fractions by mitochondria. D, the effects of ponatinib on apoptosis-related proteins.

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related proteins. Western blotting analysis in both by immunoblotting. The inhibition of the ubiquitin pro- mast cell lines indicated a decrease in levels of Mcl-1 teasome pathway resulted in the accumulation of Mcl-1 in and Bcl-2 in a dose- and time-dependent fashion, with the absence of ponatinib (Fig. 4C). However, it did not no alteration in levels of Bcl-XL,Bax,Bim,orSurvivin abrogate the ponatinib-mediated decrease of Mcl-1, sug- (Fig. 3D). gesting that the downregulation of Mcl-1 was proteasome pathway independent. Mcl-1 plays a significant role in ponatinib-induced Upon activation of caspase-3 in the apoptotic process apoptosis induced by chemotherapeutic agents, Mcl-1 is cleaved To define the significance of Mcl-1 during apoptosis into two fragments: 28 kDa Mcl-1128-350 and 17 kDa Mcl- induced by ponatinib, HMC-1.2 cells were transfected 11-127 (34). In contrast to the precursor p42 Mcl-1, p28 with empty vector or human Mcl-1 plasmid, followed Mcl-1128-350 displays a proapoptotic function (34). To by treatment of ponatinib. Ectopic overexpression of further characterize the effect of caspase-3 on Mcl-1, we Mcl-1 significantly attenuated the ponatinib-induced exposed mast cells to ponatinib with or without Z- apoptosis as reflected by the specific cleavage of PARP DEVD-fmk (a specific inhibitor of caspase-3). Western and the cell death assayed by Trypan blue exclusion blotting analysis showed that Z-DEVD-fmk did not (Fig.4A).Inaseparateapproach,HMC-1.2cellstrans- block the cleavage of ponatinib-mediated Mcl-1 (Fig. fected with mock or Mcl-1 siRNA were exposed to the 4D), although the activity of caspase-3 was inhibited, accelerating concentrations of ponatinib for 18 hours. implying that the ponatinib-mediated decrease in Mcl-1 The data showed that silencing Mcl-1 alone was not was caspase-3 independent, and might be via a yet sufficient to induce apoptosis. However, knockdown of unknown mechanism. Mcl-1 astonishingly increased the sensitivity of HMC- 1.2 cells to the ponatinib-induced apoptosis (Fig. 4A). Ponatinib abrogates the Wnt/b-catenin pathway in Our findings were consistent with the report that silenc- mast cells harboring D816V KIT ing Mcl-1 by oligonucleotides resulted in reduced sur- The Wnt/b-catenin pathway promotes the hemato- vival and increased apoptosis, and showed synergism poietic malignancy (35). Tyrosine residue phosphory- with PKC412 in HMC-1.2 cells (33). Therefore, these lation at Y654 of b-catenin by tyrosine kinases (e.g., Bcr- results indicated that Mcl-1 played a significant role in Abl, KIT, Flt3, and Src) facilitates its activity to start its ponatinib-induced apoptosis in HMC-1.2 cells. downstream target genes (e.g., c-MYC, CCND1,and AXIN2) in leukemia cells (15, 36–39). We evaluated the Ponatinib elicits Mcl-1 downregulation through impact of ponatinib on b-catenin and its effector mole- accelerating turnover rate cules. The Western blotting analysis showed a dose- and To investigate the mechanism that ponatinib dec- time-dependent decrease in total level of b-catenin in reased Mcl-1, we first ascertained whether ponatinib ponatinib-treated cells (Fig. 5A). Because the stability of accelerated the turnover of Mcl-1 protein. To this end, b-catenin is tightly regulated by GSK3b in the canonical HMC-1.2 cells were exposed to ponatinib or control Wnt/b-catenin signaling pathway, we examined and medium for 12 hours, followed by the addition of discovered that ponatinib inhibited the phosphoryla- cycloheximide and continued incubation for the indi- tion at S9 in GSK3b (inactive GSK3b) without changing cated durations. The chase-experiment results indicated its phosphorylation at Y216 (active GSK3b;Fig.5A; an accelerated turnover rate in Mcl-1 after ponatinib ref. 40). Therefore, the ponatinib-mediated decline in treatment (Fig. 4B). b-catenin was unlikely to be through GSK3b.This The degradation of many intracellular proteins in mam- notion was further supported by the finding that pro- malian cells is attributed to the ubiquitin proteasome teasome inhibitor MG132 did not reverse the ponatinib- pathway. We therefore investigated whether the acceler- mediated decline in b-catenin (data not shown). Ty- ated turnover of Mcl-1 was involved in this pathway. Cells rosine kinase Bcr-Abl was reported to control the degree were incubated with ponatinib with or without the pro- of b-catenin protein stabilization in leukemia cells (36). teasome inhibitor MG132, and then Mcl-1 was measured It remains elusive whether there is similar interaction

Figure 5. The effect of ponatinib on b-catenin signaling. A, ponatinib decreased the level of proteins involved in the b-catenin signaling pathway. B, ponatinib accelerated the turnover rate of b-catenin. After incubating with 0.3 mmol/L ponatinib for 12 hours, 0.5 mg/mL of cycloheximide (CHX) was added to HMC-1.2 cells for another few hours as indicated. Cell lysates were harvested and immunoblotted with the indicated antibodies. Data shown are one representative Western blotting results and the integrated optical density analysis by ImageJ software from three independent experiments. , P < 0.05; , P < 0.01, comparisons were made by the t test between points A and A0,BandB0. C, ponatinib decreased the levels of b-catenin both in cytoplasm and nucleus. D, ponatinib blocked the translocation of b-catenin from cytoplasm to nucleus. Photos of immunofluorescence are shown. E, ponatinib inhibited the TCF–DNA binding. Data of the EMSA assay are shown. F, ponatinib inhibited the TCF4 reporter. HMC-1.2 cells were cotransfected with plasmids of pTOPflash (or pFOPflash) and Renilla and then cells were treated with the indicated concentrations of ponatinib for 24 hours, after which cells were harvested and underwent dual luciferase reporter assay. G, b-catenin was critical in the ponatinib-induced apoptosis. HMC-1.2 cells were transfected with plasmid (empty vector or b-catenin) or siRNA (mock or b-catenin), respectively, treated with or without ponatinib and subjected to the cell death analysis (Trypan blue exclusion) or immunoblotting with the indicated antibodies.

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between KIT and b-catenin. We did notice a concomi- ing of TCF to DNA. HMC-1.2 cells were incubated with tant reduction in the phosphorylation at Y654 of b-cate- various concentrations of ponatinib for 24 hours or a nin in the ponatinib-treated cells (Fig. 5A). fixed concentration for various durations. The nuclear fractions were extracted, electrophoresed, and detected Ponatinib downregulates the expression of b-catenin with a TCF probe. Ponatinib induced a dose- and time- by accelerating its turnover rate dependent decline in levels of the TCF–DNA complex To unveil the underlying regulation of ponatinib in (Fig. 5E). b-catenin, HMC-1.2 cells were incubated with ponatinib for 12 hours, followed by the addition of cycloheximide to Ponatinib represses TCF-dependent reporter gene the cells for different durations. The expression levels of transcription b-catenin were detected by Western blotting analysis. The To verify the effect of ponatinib on the TCF/LEF tran- data showed that ponatinib accelerated the turnover of scriptional function, we performed a dual luciferase b-catenin (Fig. 5B). reporter assay. HMC-1.2 cells were cotransfected with plasmids of pTOPflash (or pFOPflash) and Renilla lucif- Ponatinib decreases b-catenin both in cytoplasm and erase, exposed to increasing concentrations of ponatinib, nucleus and subjected to the dual luciferase reporter assay. The The change in total b-catenin in ponatinib-treated data showed that ponatinib significantly reduced the cells prompted us to examine whether ponatinib affect- luciferase activity relative to control cells (Fig. 5F). ed the subcellular distribution of b-catenin. Cytoplasmic Taken together, ponatinib inhibited the b-catenin sig- and nuclear fractions of mast cells treated with ponatinib naling pathway, blocked the translocation of b-catenin, were obtained for Western blotting analysis. The results disrupted TCF–DNA binding, and suppressed the TCF/ showed that ponatinib remarkably reduced b-catenin LEF transcriptional function. both in the cytoplasmic and nuclear fractions (Fig. 5C). This effect of ponatinib was further confirmed by immu- b-Catenin is critical in the ponatinib-mediated nofluorescence microscopy (Fig. 5D). To quantitatively apoptosis measure this effect, we analyzed the intracellular activat- To examine the relative contribution of b-catenin in ed b-catenin in cells with flow cytometry and found that ponatinib-induced apoptosis, HMC-1.2 cells transfected thepercentageofABC(active-b-catenin)-positive cells with empty vector or b-catenin plasmid were treated was significantly decreased (from 46% to 23%, control with control culture medium or ponatinib for 24 hours. vs. ponatinib, respectively; Supplementary Fig. S2A). The cells transfected with empty vector underwent ex- tensive apoptosis, whereas b-catenin–transfected cells Ponatinib inhibits expression of b-catenin exhibited no increase in apoptosis, as indicated by downstream targets specific cleavage of PARP, activation of caspase-3, and Consistent with the decline of b-catenin, the expression Trypan blue exclusion (Fig. 5G). Conversely, siRNA of its key downstream targets such as c-Myc and cyclin D1 silencing of b-catenin greatly potentiated the capability was also decreased in the ponatinib-treated cells (Fig. 5A). of ponatinib to induce apoptosis. However, silencing of Furthermore, we confirmed this effect by measuring the b-catenin alone was not sufficient to trigger apoptosis. expression of AXIN2, which is believed to be a relatively Therefore, b-catenin might play an important role in exclusive target gene of b-catenin (41). The qRT-PCR apoptosis induced by ponatinib. analysis indicated that ponatinib dose-dependently decreased the mRNA levels of AXIN2 (Supplementary Ponatinib curbs the growth of xenografted HMC-1.2 Fig. S2B). cells in nude mice We assessed the in vivo effect of ponatinib with a nude Ponatinib inhibits DNA binding of TCF mouse xenograft model. The tumor growth curves (the A principal function of b-catenin in cancer lies in its estimated tumor size calculated from the tumor dimen- ability to bind TCF/LEF family transcription factors to sion over time) were significantly inhibited by ponatinib activate gene expression (42). We employed EMSA to (Fig. 6A). The tumors did not regrow during the drug evaluate the inhibitory effect of ponatinib on the bind- administration (Fig. 6A). The weights of tumors were

Figure 6. Ponatinib inhibits the growth of subcutaneous xenografts in nude mice and prolongs the survival of ASM/MCL mice. A, the estimated tumor volumes were plotted against the days of treatment. , P < 0.0001; Student t test, compared between the last measurement of the 2 groups. B, weights of tumors dissected on day 23 after inoculation of HMC-1.2 cells. , P < 0.0001; Student t test. A photograph of representative tumors is shown. C, in vivo KIT signaling was abolished by ponatinib in the subcutaneous xenografts. Western blotting results of xenograft tissues with the indicated antibodies are shown. D, the Kaplan–Meier survival curves of ASM/MCL mice treated with 20 mg/kg ponatinib are shown. E, morphologic characteristics of representative spleens and livers from normal mice and those treated with ponatinib on day 10 after injection of P815 cells. Bar charts of spleen (or liver) weight are shown. F, mononuclear cells obtained from the spleens were subjected to ﬂow cytometry analysis for expression of the pan-leukocyte antigens CD45 and CD117 as markers of mast cells. , P < 0.0001. G, the inﬁltration of mast cells in the liver and spleen was detected by hematoxylin–eosin staining. H, lysates from mononuclear cells were analyzed by immunoblotting with the indicated antibodies.

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Jin et al.

significantly lower in the treated group than in the placebo Discussion group (Fig. 6B). The body weights of mice were stable, Previous studies have shown in vitro activity of pona- with no significant difference between the placebo and tinib against tyrosine kinases such as Bcr-Abl, FIP1L1- treatment groups (data not shown). In addition, immu- PDGFRa, KIT Y823D, and FGFR1 (22). Here, we vali- nohistochemical staining showed that the levels of p-KIT, in vivo in vitro b dated the and activity of ponatinib against -catenin, and Mcl-1 were appreciably decreased in the human mast cells to support further clinical evaluation xenografted tumor tissues from ponatinib-treated mice of ponatinib in imatinib-resistant systemic mastocytosis (Supplementary Fig. S3). In addition, the decrease of Ki67 patients. staining revealed that ponatinib prominently depressed Oncogenic KIT has been reported to promote mast cell the proliferation of xenografted HMC-1.2 cells (Supple- growth via downstream targets (e.g., Stat3/5, Akt, and mentary Fig. S3). in vivo Erk1/2; ref. 12). Abrogation of PI3K signaling cascade was We further detected the effect of ponatinib on found to critically impair the transformation capability of KIT and its downstream molecules. Western blotting b cells bearing D816V KIT (12). Our results demonstrate that analysis indicated that the levels of -catenin, p-KIT, and ponatinib decreases the phosphorylation of KIT at tyro- its downstream signaling were extensively decreased in sine 568/570 residues and its downstream signaling cru- ponatinib-treated tumors (Fig. 6C). In aggregate, the cial to cell proliferation and survival, including Stat3/5, results showed that ponatinib inhibited xenografted ima- in vivo Akt, and Erk1/2. Furthermore, ponatinib induces com- tinib-resistant HMC-1.2 cells . parable cell death rates in the two cell lines as analyzed by Ponatinib prolonged the survival of mice with flow cytometry. aggressive systemic mastocytosis or mast cell Interestingly, two bands were observed when doing leukemia immunoblotting with cyclin D1 antibody in ponatinib- Injection of murine mastocytoma cell line P815 carrying treated cells (Fig. 5A). The lower bands became stronger, D814Y KIT into syngeneic DBA/2 mice is a useful model coinciding with the kinetic apoptosis after treatment, for testing potential drugs against D816V KIT (the homol- which prompted one to speculate that cyclin D1 might ogous D814Y mutation). We employed this model to eval- have been cleaved by caspase(s). However, this hypoth- uate the efficacy of ponatinib against D816V KIT. P815 cells esis will need testing in the future. (1 106) were injected into the vena caudalis of DBA/2 Ponatinib inhibited the growth of D816V KIT expres- mice. The next day, mice were randomized into two sing cells in culture, and nude mouse xenografted tumor groups: placebo [dimethyl sulfoxide (DMSO)-containing without tumor regrowth during the administration of the drug. This was further supported by the lowered expres- medium] and ponatinib (20 mg/kg, oral gavage once b daily). A group of healthy mice was also examined as a sion in Ki67, phospho-KIT, Mcl-1, and -catenin in the normal control. The survival was monitored for 20 days immunohistochemistry examination of paraffin sections after cell injection and analyzed by the Kaplan–Meier from explanted tumors. survival curve (Fig. 6D). The median survival was 10 and Mcl-1 is a prosurvival protein (43) that promotes the 18 days in mice treated with placebo and ponatinib, respec- transformation of hematopoietic stem cells and increasing tively (P < 0.001, log-rank test). the resistance to chemotherapeutic agents (44). We dem- For a cross-sectional analysis, when the placebo group onstrated that silencing Mcl-1 by siRNA increases sensi- seemed moribund on day 10 after cell injection, all three tivity of D816V KIT mutation harboring HMC-1.2 mast groups of mice were sacrificed. The placebo group exhib- cell to ponatinib, whereas overexpression of Mcl-1 atten- ited a significant increase in the WBC count and decrease uated apoptosis induced by ponatinib. These results were in the platelet and RBC counts in the peripheral blood in accord with the previous reports with TKIs (e.g., specimens (Supplementary Fig. S4). Compared with the PKC412, AMN107, and imatinib; ref. 33) and agents such ponatinib group, mice in the placebo group had increased as triptolide (43) and homoharingtonine (25) in human size and weight of liver and spleen (Fig. 6E). To analyze mast cells. Our results suggest that Mcl-1 was reduced in a the infiltration of mast cells, nucleated cells obtained from ubiquitin proteasome pathway-independent and cas- spleens were analyzed by flow cytometry for the propor- pase-3–independent manner in response to ponatinib. tion of mast cells that expressed both CD45 (pan-leuko- These findings leave it an open question the mechanism cyte antigen) and CD117. As shown in Fig. 6F, mice treated by which ponatinib decreases Mcl-1. Nevertheless, we þ þ with ponatinib had a lower proportion of CD45 CD117 and others (25, 33, 43, 44) provided results supporting a cells. The decreased infiltration in the liver and spleen in critical role of Mcl-1 in ponatinib-induced apoptosis in the ponatinib-treated mice was confirmed by light micros- cells harboring D816V KIT mutation. Our results indicated that ponatinib decreased b-cate- copy (Fig. 6G). The signal pathways were also inhibited in vitro in vivo after treatment of ponatinib as indicated by Western nin and . Our findings were in line with the in vivo previous reports that TKIs such as PKC412 and imatinib blotting (Fig. 6H). In summary, our data suggest b that ponatinib may be an important candidate for treating induce reduced -catenin (45, 46). The underlying mechanism is not clear. Our results showed an accelerated patients with mastocytosis carrying the D816V KIT b mutation. turnover rate of -catenin protein, but not supporting the

1228 Mol Cancer Ther; 13(5) May 2014 Molecular Cancer Therapeutics

Imatinib-Resistant Mast Cells Are Sensitive to Ponatinib

involvement of the ubiquitin proteasome pathway. temic mastocytosis patients with D816V KIT mutation Although we noted a concomitant reduce in the phos- may be warranted. phorylation at Y654 in b-catenin in the ponatinib-treated cells, it remains to be investigated whether this effect is Disclosure of Potential Conflicts of Interest involved in the decreased stabilization in b-catenin No potential conflicts of interest were disclosed. induced by ponatinib. b Authors' Contributions Despite the unclear mechanism of lowered -catenin by Conception and design: B. Jin, J. Pan ponatinib, several lines of evidence reveals that ponatinib Development of methodology: B. Jin causes a blockade of b-catenin activity: ponatinib Acquisition of data (provided animals, acquired and managed patients, b provided facilities, etc.): B. Jin, K. Ding abolishes the tyrosine phosphorylation of KIT and -cate- Analysis and interpretation of data (e.g., statistical analysis, biostatis- nin, blocks the translocation of b-catenin, and decreases tics, computational analysis): B. Jin, J. Pan AXIN2 Writing, review, and/or revision of the manuscript: B. Jin, J. Pan the expression of target genes of Wnt signaling ( , Administrative, technical, or material support (i.e., reporting or orga- c-MYC, and CCND1), no matter which mutation the cell nizing data, constructing databases): B. Jin, J. Pan harbors. Moreover, the decrease in TCF–DNA complexes Study supervision: J. Pan and luciferase activity of pTOPflash reveals the transcriptional inhibition of ponatinib in mast cells. Furthermore, Grant Support b This study was supported by grants from the National Natural Science ectopic overexpression of -catenin rendered a resistance Fund of China (nos. 81025021, 81373434, 91213304, 90713036, and to ponatinib-induced apoptosis while silencing b-catenin U1301226; to J. Pan), the National Basic Research Program of China increased the sensitivity. The inhibition of b-catenin by (973 Program grant no. 2009CB825506; to J. Pan), the Research Foundation of Education Bureau of Guangdong Province, China (grant cxzd 1103; to J. TKIs seems a general phenomenon because PKC412 and Pan), the Research Foundation of Guangzhou Bureau of Science and imatinib have similar effects (45, 46). Technology, China (grant to J. Pan), the National Hi-Tech Research and in vitro in vivo Development Program of China (863 Program grant no. 2008AA02Z420; to The combined results of our and studies J. Pan). suggest that ponatinib exhibits antineoplastic activity The costs of publication of this article were defrayed in part by the against human mast cells harboring imatinib-sensitive payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate and -resistant KIT mutations. Considering the present this fact. findings and the approval of ponatinib by FDA for the treatment of refractory patients with chronic myeloge- Received May 16, 2013; revised January 16, 2014; accepted February 3, nous leukemia (19), a clinical trial of ponatinib for sys- 2014; published OnlineFirst February 19, 2014.

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1230 Mol Cancer Ther; 13(5) May 2014 Molecular Cancer Therapeutics

Ponatinib Induces Apoptosis in Imatinib-Resistant Human Mast Cells by Dephosphorylating Mutant D816V KIT and Silencing β -Catenin Signaling

Bei Jin, Ke Ding and Jingxuan Pan

Mol Cancer Ther 2014;13:1217-1230. Published OnlineFirst February 19, 2014.

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