Induction of B-Chronic Lymphocytic Leukemia Cell Apoptosis by Arsenic Trioxide Involves Suppression of the Phosphoinositide 3-Kinase/Akt Survival Pathway Via

Published OnlineFirst June 9, 2010; DOI: 10.1158/1078-0432.CCR-10-0072 Published OnlineFirst on August 24, 2010 as 10.1158/1078-0432.CCR-10-0072

Cancer Therapy: Preclinical Clinical Cancer See commentary p. 4311 Research Induction of B-Chronic Lymphocytic Leukemia Cell Apoptosis by Arsenic Trioxide Involves Suppression of the Phosphoinositide 3-Kinase/Akt Survival Pathway via

c-jun-NH2 Terminal Kinase Activation and PTEN Upregulation

Javier Redondo-Muñoz1, Elizabeth Escobar-Díaz1, Mercedes Hernández del Cerro1, Atanasio Pandiella3, María José Terol4, José A. García-Marco2, and Angeles García-Pardo1

Abstract Purpose: Arsenic trioxide (ATO) induces B-cell chronic lymphocytic leukemia (B-CLL) cell apoptosis in vitro. We sought to study the mechanism involved in this effect and whether ATO is suitable for combination therapies with protein kinase inhibitors. Experimental Design: B-CLL cells were isolated from the peripheral blood of 28 patients. Cell viability studies with ATO alone or in combination with kinase inhibitors were done by flow cytometry, Western blotting, and immunofluorescence analyses. Results: After 48 hours, 3 μmol/L ATO induced apoptosis (average 75%) in all B-CLL samples studied and with minimal effect on normal peripheral blood lymphocytes. Apoptosis entailed Akt and NF-κB inactivation, XIAP downregulation, and PTEN upregulation, thus implying inhibition of the phosphoinositide 3-kinase (PI3K) survival pathway. Indeed, the combination of ATO and PI3K inhibitors increased

the apoptotic effect of either agent alone. ATO also induced c-jun-NH2 terminal kinase (JNK) activation, and this was crucial and required for subsequent apoptotic events, as inhibiting JNK activity by either gene silencing or specific inhibitors prevented Akt and NF-κB inactivation, caspase activation, and mitochondrial damage. Moreover, JNK activation was the earliest response to ATO, preceding and determining reactive oxygen species production. Conclusions: We identified the mechanism involved in ATO action on B-CLL cells and show that the combination of low doses of ATO and PI3K inhibitors efficiently induces B-CLL cell death. ATO may therefore constitute an efficient treatment for B-CLL, particularly in combined therapies. Clin Cancer Res; 16(17); 4382–91. ©2010 AACR.

B-cell chronic lymphocytic leukemia (B-CLL) is charac- IgVH unmutated status) seem to be associated with poor terized by the accumulation of malignant CD5+ B lympho- response to therapy and worse prognosis (1, 2). Most cur- cytes in the peripheral blood, and their progressive rent treatments for B-CLL consist in the administration of infiltration of lymphoid tissues (1, 2). The course of the the purine analogue fludarabine, either alone or in combi- disease is very heterogeneous, and several molecular mar- nation with other therapeutic drugs (3, 4). Although many kers (high CD38 or/and ZAP-70 expression, del17p13, patients respond well to these protocols and complete re- mission can be attained, patients eventually relapse and may die from the disease. It is therefore crucial to continue Authors' Affiliations: 1Departamento de Medicina Celular y Molecular, searching for new compounds that could be useful in the Centro de Investigaciones Biológicas, Consejo Superior de treatment of this type of leukemia, especially in the ad- Investigaciones Científicas and 2Servicio de Hematología, Hospital Universitario Puerta de Hierro, Madrid, Spain; 3Centro de Investigación vanced setting. del Cáncer, CSIC-Universidad de Salamanca, Salamanca, Spain; and Arsenic trioxide (ATO) has been used in traditional 4Departamento de Hematología y Oncología Médica, Hospital Clínico, Valencia, Spain Chinese medicine and is currently successfully employed for the treatment of acute promyelocytic leukemia (APL; Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). refs. 5–8). ATO is also being trialed for other hematologic J. Redondo-Muñoz and E. Escobar-Díaz contributed equally to this work. malignancies, including chronic myeloid leukemia, mye- lodysplastic syndrome, and non-M3 acute myelocytic leu- Corresponding Author: Angeles García-Pardo, Centro de Investigaciones Biológicas, Ramiro de Maeztu 9, 28040 Madrid, Spain. Phone: 34-91-837- kemia and multiple myeloma, either as monotherapy or 3112; Fax: 34-91-536-0432; E-mail: [email protected]. combined therapy (7, 9). The cytotoxic effect of ATO on doi: 10.1158/1078-0432.CCR-10-0072 these systems involves complex mechanisms that are not ©2010 American Association for Cancer Research. fully understood. In many cases, induction of apoptosis

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was >95% CD19+, >70% CD5+, considered suitable for Translational Relevance subsequent studies. Purified B-CLL cells were either used immediately or frozen and stored in liquid nitrogen. B-cell chronic lymphocytic leukemia (B-CLL) re- Peripheral blood lymphocytes (PBL; T plus B lympho- mains an incurable disease, making it crucial to con- cytes) from five adult healthy donors were obtained from tinue searching for novel therapies. Arsenic trioxide buffy coat preparations routinely used for monocytic cell (ATO) is an efficient treatment for acute promyelocytic purification. After Ficoll-Hypaque centrifugation, mono- leukemia, and it is being trialed for other hematologic cytic cells were removed using anti-CD14-conjugated malignancies. To explore the potential clinical use of microbeads and MACS separation columns (Miltenyi ATO in B-CLL we studied the mechanism by which Biotec). B-CLL and control cells were cultured at 1.5 × ATO induces apoptosis in B-CLL cells. We report that 106/mL in RPMI/10% fetal bovine serum, 100 μg/mL JNK and PTEN are crucial molecules and inhibit phos- gentamicin and penicillin, and 100 U/mL streptomycin phoinositide 3-kinase (PI3K) survival signaling in (Invitrogen Ltd.). response to ATO. Accordingly, PI3K inhibitors effi- Approval was obtained from the Consejo Superior de ciently enhanced the apoptotic effect of ATO. These ef- Investigaciones Científicas Bioethics Review Board for fects were observed at low, nontoxic ATO doses and in these studies. all cases studied. Our results strongly suggest that ATO alone or combined with PI3K inhibitors may be con- RNA interference experiments sidered an alternative therapy for B-CLL. The small interfering RNA (siRNA) sequence targeting JNK: sense 5′-CAAAGAUCCCUGACAAGCAdTT-3′ (s11153), and the control siRNA sequence: sense 5′-AUU- ′ by ATO involves the release of reactive oxygen species GUAUGCGAUCGCAGACdTT-3 , were custom-made (ROS), and experimental reduction of glutathione peroxi- by Ambion. For transfection of B-CLL cells, siRNAs μ dase increases the sensitivity to ATO (10, 11). Blocking (500 nmol/L final concentration) in 100 LRPMIwere the extracellular signal-regulated kinase (ERK)/ERK, phos- incubated at room temperature for 10 minutes with – 4.5 μL HiPerfect transfection reagent (Qiagen). The mixture phoinositide 3-kinase (PI3K)/Akt, or p38 mitogen-activated 6 protein kinase signaling pathways has also been shown to was added dropwise to 2 × 10 cells in RPMI/10% FCS, and enhance the apoptotic effect of ATO on APL, myeloma, transfected cells were used after 24 hours. The efficiency of and T-leukemia cells (10, 12–15). Additionally, ATO- gene silencing was analyzed by Western blotting. induced apoptosis of myeloma and APL cells involves the activation of c-jun-NH-kinase (JNK; refs. 16, 17). Analysis of apoptosis and mitochondrial membrane Δψ Very few studies have addressed the effect of ATO on potential ( m) 5 μ B-CLL cells. In analyses of a small number of B-CLL sam- B-CLL cells (1.5 × 10 ) in 100 L RPMI/10% fetal ples (18) or long exposure times (19), ATO was shown bovine serum were placed in 96-well plates and treated with various concentrations of ATO or vehicle (PBS). After to induce apoptosis of B-CLL cells, concomitant with 6 downregulation of Bcl-2. ATO also induced ROS genera- 24 to 48 hours cells were suspended (10 /mL) in 1× bind- μ tion and significantly enhanced the cytotoxic activity of ing buffer (Bender Medsystems), containing either 3 L μ 2-methoxyestradiol and ascorbic acid on B-CLL samples FITC-Annexin V and 50 g propidium iodide (PI), or (20, 21). It was recently reported that ATO preferentially PI alone. After 10 minutes, cells were analyzed by flow induced apoptosis in B-CLL cases with unfavorable prog- cytometry on a Coulter Epics XL. For combined treat- nosis (22). These previous studies strongly suggest that ments, B-CLL cells were incubated for 30 minutes with μ ATO, alone or in combination with other treatments, either SP600125 (10 mol/L), SB203580, BisI, UO126, μ could be an efficient therapeutic agent for B-CLL. The Tricirbine/API-2, LY294002 (all at 5 mol/L), Z-VAD- μ mechanism by which ATO induces B-CLL cell apoptosis FMK, or Z-IETD-FMK (both at 50 mol/L) prior to the ad- μ Δψ has not been established, and we have addressed this in dition of 2 mol/L ATO. For m measurements, B-CLL the present report. cells were incubated for 24 hours with or without 3 μmol/L ATO and treated for 20 minutes with 20 nmol/L DiOC6 (Cabiochem) at room temperature in the dark. Cells Materials and Methods were washed, resuspended in PBS, and analyzed by flow cytometry. Patients, B-CLL cell purification, and normal peripheral blood lymphocytes Measurement of intracellular ROS accumulation Following informed consent, B-CLL cells were purified B-CLL cells (5 × 105) were treated with or without from the peripheral blood of 28 patients (Table 1) by SP600125 (2-10 μmol/L), NAC (10 mmol/L), or MnTBAP Ficoll-Hypaque (Nycomed) centrifugation and anti-CD3- (50 μmol/L) for 30 minutes and cultured in the presence conjugated Dynabeads (Dynal Biotech ASA). Myeloid cells or absence of 3 μmol/L ATO. After 16 hours, 5 μmol/L of were not removed by these procedures, but they represent H2DCFDA were added and fluorescence was analyzed a minor contamination, as the resulting cell population after 30 minutes by flow cytometry.

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Table 1. Clinical characteristics of B-CLL patients

Patients Sex/Age Stage* ZAP70/CD38† Ig status Genetic alterations

P1 M/76 B/II ND/+ Mutated del13q14 P2 F/35 B/II ND/− Mutated del13q14 P3 M/62 B/II +/− Unmutated del13q14 P4 F/73 A/0 +/− Mutated del13q14, Trisomy 12 P5 M/56 A/I +/+ ND del13q14 P6 F/73 A/0 +/+ Mutated Trisomy 12 P7 M/64 A/0 ND/− ND No P8 M/42 C/IV +/− ND No P9 F/80 B/II −/− Mutated T 14;19 P10 M/65 B/II +/− Mutated del13q14 P11 F/63 A/0 −/− ND del13q14, del17p13 P12 F/72 B/II ND/+ Unmutated Trisomy 12 P13 M/47 A/0 −/− Mutated No P14 M/53 C/III ND/− ND del13q14 P15 F/78 A/0 −/− Unmutated No P16 M/63 B/II +/+ Mutated No P17 F/59 A/0 +/− Unmutated No P18 M/74 A/0 −/− ND del13q14 P19 F/81 A/0 −/− Mutated No P20 M/54 A/0 ND/ND Unmutated ND P21 M/82 A/0 −/− Unmutated del17p13 P22 F/75 A/0 ND/+ Mutated del13q14 P23 M/54 B/II +/+ Unmutated No P24 F/71 A/0 −/− Unmutated del13q14 P25 M/78 B/II ND/− Unmutated Trisomy 12 P26 M/60 A/0 −/− Mutated del13q14 P27 M/55 A/II +/+ Unmutated del13q14 P28 F/71 B/II +/+ Mutated del17p13

Abbreviation: ND, not determined. *According to Rai et al. (40) and Binet et al. (41). †The CD38 and ZAP70 expression in B-CLL cells has prognostic value (1, 2). None of the patients had received previous treatment at the time of this study.

Preparation of nuclear and cytosolic extracts and equal amounts from both fractions were analyzed by B-CLL cells (20 × 106) with or without previous incuba- SDS-PAGE and Western blotting. tion (2 hours, 37°C) with 3 μmol/L ATO were harvested, Additional information may be found in Supplemen- washed, and resuspended in 400 μL of cold buffer A tary Materials. [10 mmol/L HEPES (pH 7.6), 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 0.75 mmol/L spermi- Results dine, 0.75 mmol/L spermine, 1 mmol/L DTT] with 1 mmol/L MoO4Na2, 1 mmol/L NaF, and protease inhibi- ATO induces apoptosis of B-CLL cells but not of tors (aprotinin, leupeptin, pepstatin, phenylmethylsulfo- normal PBL nylfluoride). After 15 minutes on ice, 10% Nonidet-P40 To establish the best conditions for studying ATO effect, was added and lysates were vortexed for 10 seconds and B-CLL cells (12 patients) were incubated in RPMI/10% fe- centrifuged. The supernatant (cytosolic fraction) was re- tal bovine serum with several doses of ATO or vehicle, and moved and 50 μL of cold buffer C [20 mmol/L HEPES their viability was measured by flow cytometry using (pH 7.6), 400 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L PI. Figure 1A shows that ATO significantly induced apo- EGTA, protease inhibitors) were added to the pellet ptosis in a dose-dependent manner, confirming previous (nuclear fraction) and incubated on a rotary shaker reports (19–22). All samples tested were sensitive to (4°C, 20 minutes). After centrifugation, protein content ATO, independently of the clinical stage, the ZAP-70/ was determined by the bicinchoninic acid assay (Pierce), CD38 expression, or whether they had been frozen or were

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freshly isolated. At 3 μmol/L, a concentration within the the PI3K/Akt inhibitors LY294002 or API-2 produced range used for APL treatment (8, 16), ATO reduced limited, albeit significant, apoptosis (33-38%, average of 4 B-CLL cell viability by 50% and 75% after 24 and 48 hours, samples) after 48 hours, compared with untreated cells. respectively, compared with untreated cells, which re- However, when ATO was combined with either inhibitor, mained highly viable under these culture conditions apoptosis significantly increased to 68% and 77% for (Fig. 1A). In contrast, 3 μmol/L ATO had a minor effect LY294002 and API-2, respectively (Fig. 2A). In contrast, (15% cell death after 48 hours) on normal PBL the PKC inhibitor BisI had no effect on B-CLL cell viability (Fig. 1B). At higher doses, ATO induced 90% B-CLL cell and did not increase the apoptotic effect of ATO (Fig. 2A). death, but started to produce a significant effect on normal To then determine whether ATO-induced apoptosis af- PBL (Fig. 1B). Subsequent experiments were done using fected the PI3K signaling pathway, we measured Akt phos- 3 μmol/L ATO except when combined with other agents phorylation and expression of the Akt targets NF-κB and in which 1 to 2 μmol/L ATO was used. XIAP (25, 26). As shown in Fig. 2B, for two representative patients Akt was constitutively phosphorylated, and phos- ATO cooperates with PI3K/Akt inhibitors to enhance phorylation significantly decreased after 3 hours of ATO B-CLL cell apoptosis and inhibits Akt activation by exposure, being almost undetectable after 24 hours. upregulating PTEN Phospho-Akt remained unaffected in control cells. In cor- The PI3K/Akt and protein kinase C (PKC) signaling relation with this, the levels of the NF-κB–associated pro- pathways are constitutively activated in many B-CLL cases tein IκB-α clearly increased, indicating that it remained and contribute to their resistance to apoptosis (23, 24). bound to NF-κB in the cytoplasm, likely preventing We studied whether the combination of ATO and PI3K/ NF-κB activation. The antiapoptotic protein XIAP also sig- Akt or PKC inhibitors enhanced the apoptotic effect of either nificantly decreased upon 24 hours of ATO exposure agent alone. When used individually, ATO (2 μmol/L), or (Fig. 2B). Interestingly, and parallel to these observations, expression of the phosphatase PTEN, a well-known PI3K inhibitor (27) significantly increased (4-fold, P ≤ 0.01) after 24 hours of ATO treatment (Fig. 2B). Altogether these results indicated that ATO-induced B-CLL cell apoptosis involved PTEN upregulation and inhibition of the PI3K/ Akt/NF-κB survival pathway.

JNK plays a critical role in the induction of B-CLL cell apoptosis by ATO To determine whether ATO induction of B-CLL cell apoptosis involved JNK activation, we first measured JNK phosphorylation after 24 hours of ATO exposure. Figure 3A shows that ATO clearly induced JNK activation that was prevented by the JNK inhibitor SP600125 in a dose-dependent manner, with complete inhibition at 10 μmol/L. SP600125 had no effect in the absence of ATO (Fig. 3A). Subsequent kinetic studies revealed that JNK was already phosphorylated after 1 hour of ATO treatment, therefore preceding Akt dephosphorylation and PTEN upregulation (Figs. 2B and 3B). Indeed, JNK was responsible for these subsequent events as both were partially inhibited by 5 μmol/L SP600125 (50% and 30% inhibition for Akt and PTEN, respectively; not shown) and completely inhibited by 10 μmol/L SP600125 (Fig. 3B). Further evidence for a crucial role of JNK in ATO-induced B-CLL cell apoptosis was obtained by analyzing NF-κB activation. As shown in Supplementary Fig. S1A, and in agreement with the data shown in Fig. 2B for IκB-α,NF-κB nuclear levels were clearly diminished by ATO but were unaffected by the ATO/SP600125 combination. More- over, modulation of NF-κB activity in correlation with Fig. 1. Effect of ATO on B-CLL cell viability. A, B-CLL cells were PTEN expression was apparently specific for JNK because incubated with the indicated concentrations of ATO or vehicle. After 24 or the PI3K inhibitor LY294002, which efficiently induced 48 hours, cell viability was determined by flow cytometry using PI. κ α B, PBL from five different healthy individuals were analyzed as above. Akt dephosphorylation and I B- upregulation, had no Determinations were done in duplicate and average values ± SD are effect on PTEN, either alone or combined with ATO shown. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.0001. (Supplementary Fig. S1B).

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To confirm the above results, we transfected B-CLL cells Fig. S2B), indicating that both the extrinsic and intrinsic with JNK siRNA and measured viability, Akt inactivation, (mitochondrial) apoptotic pathways were activated by and PTEN expression. Figure 3C shows that JNK siRNA re- ATO in B-CLL cells. Importantly, all these apoptotic events duced JNK levels by 78% resulting in complete inhibition were completely prevented by inhibiting JNK (Supplemen- of the ATO effect on Akt and PTEN (Fig. 3C). Transfection tary Fig. S2B). with control siRNA, however, did not affect the cell re- To further assess the mitochondrial involvement in sponse to ATO (Fig. 3C). In correlation with these results, ATO-induced B-CLL cell apoptosis, we first studied whe- SP600125 or JNK siRNA transfection inhibited the apo- ther ATO induced mitochondrial depolarization. B-CLL ptotic effect of ATO, but had no effect in the absence of cells from two patients were incubated for 24 hours in this agent (Fig. 3D). the absence or presence of 3 μmol/L ATO, treated with In agreement with recent reports (21, 22), induction of DiOC6, and analyzed by flow cytometry. Figure 4A shows B-CLL cell apoptosis by ATO was mediated by caspases, as that ATO decreased the mitochondrial membrane poten- the pan caspase inhibitor Z-VAD-FMK or the caspase-8 in- tial, increasing the number of DiOC6-negative, apoptotic hibitor Z-IETD-FMK nearly completely abrogated the ATO cells in both cases. Next, we studied cytochrome c release apoptotic effect (samples, n = 5; Supplementary Fig. S2A). by confocal microscopy using the mitochondrial protein Indeed, ATO induced caspase-9, -8, and -3 activation and Hsp60 as reference. Figure 4B shows for a representative cleavage of their specific substrates (Supplementary sample that after 24 hours, a clear colocalization of

Fig. 2. ATO interferes with the PI3K/Akt survival pathway. A, B-CLL cells (4 patients) were incubated for 30 minutes with or without 5 μmol/L LY294002, API-2, or BisI, prior to the addition of 2 μmol/L ATO. After 48 hours, cell viability was analyzed by flow cytometry. Const. (constitutive), fresh cell viability. B, B-CLL cells treated or not with 3 μmol/L ATO were lysed at different times, and protein expression was analyzed by Western blotting. Normalized average values are shown. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.0001.

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Fig. 3. JNK is activated by ATO in B-CLL cells and influences the PI3K/Akt pathway and PTEN expression. A, B-CLL cells, treated or not with SP600125 (SP, 30 minutes), were incubated with or without 3 μmol/L ATO. Cells were lysed after 24 hours and analyzed by Western blotting. B, B-CLL cells treated or not with 10 μmol/L SP for 30 minutes were incubated with or without 3 μmol/L ATO. Cells were lysed at the indicated times and analyzed by Western blotting. C, B-CLL cells transfected with control (Ctl) or JNK siRNA were treated with or without 3 μmol/L ATO. After 24 hours, cells were lysed and analyzed by Western blotting. D, B-CLL cells, treated with SP600125 or transfected with control or JNK siRNA were cultured with or without 3 μmol/L ATO for 48 hours. Viability was measured by flow cytometry using Annexin V and PI. Const. (constitutive), fresh cell viability. ***, P ≤ 0.0001.

cytochrome c and Hsp60 was observed on control cells but was not affected by ZVAD-FMK or Z-IETD-FMK, indicating not on ATO-treated cells, confirming the cytochrome c mi- that it preceded caspase activation (data not shown). tochondrial release. As observed for caspase activation Generation of ROS mediates ATO-induced apoptosis in (Supplementary Fig. S2B), cytochrome c release was com- many cell types and apparently plays an essential function pletely prevented by inhibiting JNK signaling (Fig. 4B). in the case of B-CLL cells (21, 22). We therefore studied Accordingly, active Bax, a proapoptotic factor involved in whether JNK activity was required for induction of ROS the mitochondrial pathway, was readily detected on ATO- in response to ATO. B-CLL cells were treated with treated cells, but was absent on control cells or in the pres- 3 μmol/L ATO, and ROS levels were measured using the ence of the JNK inhibitor (Fig. 4B). Cytochrome c release fluorescent probe H2DCFDA. Figure 4C shows that ROS

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Fig. 4. JNK activation is critical for the ATO apoptotic effect in B-CLL cells. A, flow cytometric analysis of the loss of mitochondrial membrane potential

(Δψm) after B-CLL cell incubation with 3 μmol/L ATO for 24 hours. B, B-CLL cells treated or not with 10 μmol/L SP for 30 minutes were cultured with or without 3 μmol/L ATO. After 24 hours, cells were analyzed by confocal microscopy using antibodies for cytochrome c, or active Bax (green) and Hsp60 (red). Colocalization of cytochrome c and Hsp60 is further shown by dot-plot analyses. C, B-CLL cells treated or not with SP or transfected with

control (Ctl) or JNK siRNA were incubated with or without ATO for 16 hours. H2DCFDA was added, and ROS production was measured by flow cytometry. Shaded areas, untreated cells; green lines, 10 μmol/L SP-treated cells; blue lines, ATO-treated cells; red lines, SP/ATO-treated cells. Average mean fluorescence intensity values are shown. D, B-CLL cells were treated with 10 mmol/L NAC or 50 μmol/L MnTBAP and cultured with or without 3 μmol/L ATO

for 24 hours. H2DCFDA was added, and ROS production was measured by flow cytometry. Shaded areas, untreated cells; green lines, cells treated with NAC or MnTBAP; blue lines, ATO-treated cells; red lines, NAC/ATO- or MnTBAP/ATO-treated cells. Quantitative values are also shown. In parallel, cells were lysed and JNK activation was analyzed by Western blotting. **, P ≤ 0.01.

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production was significantly increased (P ≤ 0.01) after upregulation and PI3K/Akt inactivation, and (c)ATO 16 hours of ATO exposure, compared with control cells. cooperates with PI3K/Akt inhibitors enhancing B-CLL cell The JNK inhibitor SP600125 had no effect when added apoptosis. alone, but prevented the ATO-induced ROS production All 28 patient samples analyzed were very sensitive to in a dose-dependent manner (Fig. 4C). Accordingly, JNK the cytotoxic effect of ATO, regardless of their prognostic gene silencing also inhibited ROS production in response markers or clinical stage. This included two B-CLL samples to ATO, whereas the control siRNA had no effect (Fig. 4C). carrying a deletion in chromosome 17p13 (which confers To then establish whether ROS induction preceded or drug resistance), in agreement with a previous report (22). followed JNK activation, we used the antioxidants Importantly, efficient B-CLL cell apoptosis was attained by N-acetylcysteine (NAC) and Mn-tetrakis-(4-benzoic acid) concentrations of ATO (3 μmol/L) currently used in the porphyrin (MnTBAP). Incubation of B-CLL cells with clinical treatment of APL (8, 16), with minimal effect on NAC or MnTBAP prior to ATO treatment completely pre- normal PBL, further emphasizing the potential therapeutic vented the increased ROS production (Fig. 4D) and the use of ATO in B-CLL. ATO apoptotic effect, yielding 55% and 54% viable cells The PI3K/Akt signaling pathway is constitutively activa- for NAC and MnTBAP, respectively, compared with 13% ted in many B-CLL cases, as we also find in our study, and (no antioxidant) and 68% (control; not shown). In con- contributes to the typical B-CLL cell antiapoptotic pheno- trast, NAC or MnTBAP had no effect on the levels of JNK type (23, 24). In this report we show for the first time that phosphorylation, which remained elevated (Fig. 4D). suboptimal concentrations of ATO (2 μmol/L) specifically Similar results were obtained with catalase (not shown). cooperated with pharmacologic inhibitors of the PI3K sur- Altogether these results established that JNK activation is vival pathway and enhanced B-CLL cell apoptosis. Low an early B-CLL cell response to ATO and is crucial for doses of ATO may thus prove to be useful in combined subsequent events that lead to apoptosis. Figure 5 shows therapies with PI3K inhibitors. Our study also shows that a schematic representation of the ATO-induced apoptotic induction of B-CLL cell apoptosis by ATO was accompa- mechanism in B-CLL cells. nied by inhibition of the PI3K/Akt signaling pathway, assessed by NF-κB inactivation and downregulation of Discussion the Akt (26) and NF-κB (28) target XIAP. Modulation of NF-κBactivitybyATOseemstobespecificasother We studied the mechanism involved in induction of antiapoptotic proteins such as Mcl-1, which is mainly B-CLL cell apoptosis by ATO and whether ATO is suitable regulated by signal transducers and activators of transcrip- for combination therapies with protein kinase inhibitors. tion (STAT) family members (29), did not change upon We show for the first time that (a) JNK activation is critical ATO exposure (data not shown). Thus, although ATO for the ATO effect on B-CLL cells, (b) ATO induces PTEN was shown to reduce STAT activity in AML (30) and

Fig. 5. Schematic diagram showing the apoptotic mechanism induced by ATO in B-CLL cells. Left, in the absence of ATO, the PI3K/Akt/NF-κB pathway is constitutively activated, leading (among other signals) to XIAP upregulation and cell survival. Right, ATO induces JNK activation, resulting in inhibition of the PI3K/Akt/NF-κB signaling pathway. Downregulation of NF-κB and XIAP (both PTEN repressors) contribute to PTEN upregulation, also probably induced by direct JNK action (dotted lines). JNK activation finally leads to ROS production, mitochondrial damage and apoptosis.

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downregulate Mcl-1 in myeloma cells (31), our results tion upstream of ROS mostly leads to necrosis (35), our suggest that this is not the case in B-CLL. results clearly show that in B-CLL cells, JNK activation Parallel to the observed NF-κB inactivation by ATO, our preceded ROS formation and initiated the apoptotic results also show a significant upregulation of PTEN. response to ATO. NF-κB has been shown to repress PTEN expression, this Our study therefore establishes the mechanism of ATO being one of the mechanisms by which NF-κB does its action in B-CLL cells, revealing a crucial role for JNK and antiapoptotic function in tumors (32). The NF-κBtarget PTEN.Itisalsothefirsttoreportthatwhencombined XIAP was also recently shown to ubiquitinate PTEN and with PI3K inhibitors, very low doses of ATO were sufficient to regulate its content and compartmentalization (33). to produce efficient malignant cell death. The use of ATO As PTEN specifically inhibits PI3K signaling through its in mixed therapies has been proven very successful in the lipid phosphatase activity (27, 34), our findings indicate killing of myeloma, APL, and T-leukemia cells in vitro (5, 7, that ATO-induced B-CLL cell apoptosis affects the PI3K 10). As we now show, the combination of ATO and PI3K survival pathway at various levels. They also provide the inhibitors may represent a promising and efficient alterna- first evidence for a possible functional role of PTEN in tive for the clinical treatment of B-CLL. B-CLL cells. PTEN induction also decreased the resistance of HL-60 monocytic cells to the apoptotic effect of ATO Disclosure of Potential Conflicts of Interest (13, 35), again highlighting that at least in certain cells, suppression of the PI3K/Akt/NF-κB survival pathway is No potential conflicts of interest were disclosed. crucial for the ATO cytotoxic action. Acknowledgments NF-κB inactivation, however, may not be sufficient to upregulate PTEN in B-CLL cells, as our results also show We thank the B-CLL patients who donated blood samples for this that inhibiting the PI3K/Akt/NF-κB pathway in the ab- research, Dr. Patricio Aller for reviewing the manuscript and providing sence of ATO did not upregulate PTEN or enhance the ef- some reagents, and Dr. Pedro Lastres for help with the flow cytometry analyses. fect of ATO on PTEN. This suggests that other molecules specifically induced by ATO may also contribute to PTEN Grant Support upregulation. One such molecule is likely JNK. It is known that JNK activation counteracts the survival effect of NF-κB Grants PI060400, RD06/0020/0011, and SAF2009-07035 (A. García- (36), regulates PTEN expression (37), and may be a target Pardo), RD06/0020/0041 (A. Pandiella), and PI061637 and RD06/0020/ 0080 (M.J. Terol) from the Ministerio de Ciencia e Innovación, and grant of PTEN in certain cell types (38). JNK is involved in ap- from the Fundación de Investigación Médica Mutua Madrileña (A. García- optoticsignalinginmanycelltypes(39)andhasbeen Pardo). JRM was supported by a fellowship from the Fundación Ramón shown to mediate the cytotoxic effect of ATO in APL Areces, Madrid, Spain. The costs of publication of this article were defrayed in part by the (16) and myeloma (17) cell lines. As we show in this re- payment of page charges. This article must therefore be hereby marked port, JNK activation was an early response to ATO treat- advertisement in accordance with 18 U.S.C. Section 1734 solely to ment and was required for subsequent apoptotic events indicate this fact. in B-CLL cells, including PTEN upregulation and ROS pro- Received 01/12/2010; revised 04/29/2010; accepted 05/27/2010; duction. Thus, although it was suggested that JNK activa- published OnlineFirst 06/09/2010.

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Induction of B-Chronic Lymphocytic Leukemia Cell Apoptosis by Arsenic Trioxide Involves Suppression of the Phosphoinositide 3-Kinase/Akt Survival Pathway via c-jun-NH2 Terminal Kinase Activation and PTEN Upregulation

Javier Redondo-Muñoz, Elizabeth Escobar-Díaz, Mercedes Hernández del Cerro, et al.

Clin Cancer Res Published OnlineFirst June 9, 2010.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-10-0072

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