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Arsenic Trioxide (ATO) and MEK1 Inhibition Synergize to Induce Apoptosis in Acute Promyelocytic Leukemia Cells

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Arsenic Trioxide (ATO) and MEK1 Inhibition Synergize to Induce Apoptosis in Acute Promyelocytic Leukemia Cells Leukemia (2005) 19, 234–244 & 2005 Nature Publishing Group All rights reserved 0887-6924/05 $30.00 www.nature.com/leu Arsenic trioxide (ATO) and MEK1 inhibition synergize to induce apoptosis in acute promyelocytic leukemia cells P Lunghi1, A Tabilio2, F Lo-Coco3, P Pelicci4 and A Bonati1 1Department of Clinical Sciences, Section of Hemato-Oncology, University of Parma, Parma, Italy; 2Department of Clinical and Experimental Medicine, Section of Hematology and Clinical Immunology, University of Perugia, Italy; 3Department of Biopathology, University of Rome ‘Tor Vergata’, Italy; and 4Department of Experimental Oncology, Milan, Italy Recent studies suggest that components of the prosurvival inhibits cell growth and induces apoptosis through both PML- signal transduction pathways involving the Ras-mitogen-acti- RARa-dependent and -independent mechanisms with caspase vated protein kinase (MAPK) can confer an aggressive, 7–9 apoptosis-resistant phenotype to leukemia cells. In this study, activation of neoplastic cells. we report that acute promyelocytic leukemia (APL) cells exploit In vitro and in vivo studies have been conducted to evaluate the Ras-MAPK activation pathway to phosphorylate at Ser112 the effect of ATO in combination with ATRA or other biological and to inactivate the proapoptotic protein Bad, delaying arsenic compounds such as cAMP. The results showed that these trioxide (ATO)-induced apoptosis. Both in APL cell line NB4 and associations are more effective than the single agents in in APL primary blasts, the inhibition of extracellular signal- inducing differentiation or cell apoptosis.10–12 regulated kinases 1/2 (ERK1/2) and Bad phosphorylation by MEK1 inhibitors enhanced apoptosis in ATO-treated cells. We According to recent laboratory studies, the blast cells of most isolated an arsenic-resistant NB4 subline (NB4-AsR), which acute myelogenous leukemias (AML) including APL show showed stronger ERK1/2 activity (2.7-fold increase) and Bad constitutive activation of extracellular signal-regulated kinases phosphorylation (2.4-fold increase) compared to parental NB4 1/2 (ERK1/2) as well as of the kinases immediately upstream of cells in response to ATO treatment. Upon ATO exposure, both ERK, known as mitogen-activated protein (MAP)/ERK kinases R NB4 and NB4-As cell lines doubled protein levels of the death (MEKs).13–16 Furthermore, we and others have demonstrated that antagonist Bcl-xL, but the amount of free Bcl-xL that did not R downmodulation of MEK1 phosphorylation inhibits proliferation heterodimerize with Bad was 1.8-fold greater in NB4-As than in 17–20 the parental line. MEK1 inhibitors dephosphorylated Bad and and induces apoptosis of primary AML blasts (reviewed in 21 22 inhibited the ATO-induced increase of Bcl-xL, overcoming ATO Platanias and Lee and McCubrey ). In this study, we aimed at resistance in NB4-AsR. These results may provide a rationale to investigating whether the combination of ATO with agents that develop combined or sequential MEK1 inhibitors plus ATO block the phosphorylation of MEK1 can potentiate the antileu- therapy in this clinical setting. kemic action of ATO in APL. Moreover, blocking MEK1 function Leukemia (2005) 19, 234–244. doi:10.1038/sj.leu.2403585 Published online 11 November 2004 with specific inhibitors or specific double-stranded RNA Keywords: arsenic trioxide; MEK1 inhibition; acute promyelocytic oligonucleotides (siRNA) restored ATO sensitivity in an NB4 leukemia; MEK1 sIRNA; leukemic cells apoptosis; bad cell line rendered resistant to ATO-induced apoptosis. Finally, phosphorylation we demonstrated here that the downmodulation of MEK1 phosphorylation significantly enhanced ATO-induced apoptosis in primary APL blasts. Introduction Materials and methods It has been demonstrated that low doses (0.06–0.6 mg/km of body weight/day) of arsenic trioxide (ATO) are extremely Reagents efficacious in the treatment of relapsed acute promyelocytic leukemia (APL), inducing partial differentiation and promoting ATO was purchased from Sigma (St Louis, MO, USA). A 1 mmol/l apoptosis of malignant promyelocytes.1 This pilot trial con- stock solution was obtained by dissolving ATO in phosphate- ducted at the Memorial Sloan-Kettering Cancer Center together buffered saline (PBS): the solution was diluted to working with a more recent multicenter clinical study2 confirmed the concentration immediately before use. A 100 mM stock solution impressive results obtained by Chinese investigators in early of the MEK1 inhibitors PD98059 (20-amino-30-methoxyflavone; studies on ATO in the treatment of this disease.3–5 In particular, Cell Signaling Technology, Beverly, MA, USA) or PD184352 (2- the efficacy, safety and limited hematological toxicity of this [chloro-4-iodo-phenylamino]-N-cyclopropylmethoxy-3,4-di- approach was brought to light.2 Currently, ATO is considered fluoro-benzamide) kindly provided to us by Dr JS Sebolt- the treatment of choice for patients with relapsed disease, Leopold (Cancer Molecular Sciences, Pfizer Global Research & particularly in patients exposed to all-trans retinoic acid (ATRA) Development, Ann Arbor, MI, USA) was prepared in dimethyl within the prior 12 months.6 sulfoxide (DMSO). These reagents are highly selective inhibitors The mechanisms of action of ATO have been investigated 23,24 of MEK1 phosphorylation and activation. The doses used by both in vivo and in vitro: at low doses (0.1–0.25 mM), ATO us of 1 or 2 mM for PD184352 and of 10, 20 or 40 mM for induces a partial differentiation in APL cells through degradation PD98059 were those that proved effective in vitro in leukemic of PML-RARa, while at higher doses (0.5–2 mM), this agent 17–19 cells as documented both by ourselves and other authors. The doses of ATO hereby employed were selected based on the Correspondence: Professor A Bonati and Dr P Lunghi, Dipartimento di evidence that ATO, from concentrations of 0.5 to 2 mM upwards, Scienze Cliniche, Via Gramsci 14, Parma 43100, Italy; Fax: þ 39 0521 941290; E-mails: [email protected] and [email protected] has the capacity to induce apoptosis without differentiation in Received 4 March 2004; accepted 28 September 2004; Published NB4 cells. These in vitro concentrations are within the range online 11 November 2004 found in the plasma of patients receiving ATO treatment for APL MEK1 inhibition and arsenic trioxide in APL P Lunghi et al 235 and entering complete hematologic remission after such with MitoLight reagent for 20 min at 371C. After centrifugation, therapy.25 they were resuspended in incubation buffer and analyzed by fluorescence-activated cell sorter scanner. MitoLight emits a red fluorescence (MitoLight aggregates, high DCm) when seques- Cell lines and patient primary blasts tered in the mitochondrial membrane of healthy cells detectable in the PI channel. In apoptotic cells with altered mitochondrial R Selection of NB4 arsenic-resistant cell line (NB4-As ). An membrane potential, the dye in its monomeric form remains in R arsenic-resistant NB4 subline named NB4-As Paolo Lunghi 1 the cytoplasm, emitting a green fluorescence (MitoLight mono- R (PL1), hereafter referred to as NB4-As , was derived from the mers, low DC ) detectable in the fluorescein isothiocyanate 10 m NB4 cell line following the method described by Gianni et al. channel. R NB4-As was obtained by treating parental cells with ATO 1 mM weakly and was maintained with the same dose. In the presence R of high doses of ATO (1 mM), the NB4-As grew, although at a siRNA transfections reduced rate, while the parental cell line died. NB4-AsR cells R were also constantly grown in the presence of 1 mM ATO. In Prior to electroporation, NB4-As cells were washed twice with experiments examining the response of NB4-AsR to ATO, the serum-free Opti-MEM (Gibco BRL Paisley, UK) and resuspended 6 cells were first washed thoroughly to remove ATO from the to a final concentration of 8 Â 10 cells/ml in Opti-MEM (Gibco media, and then cultured for 24 h in the media alone prior to BRL). Subsequently, 0.5 ml of cell suspension was mixed either initiating the experiment. with 0.5 nmol of Smart pool double-stranded RNA oligonucleo- Human leukemia cell lines NB4 or NB4-AsR in logarithmic tides (siRNA) against MEK1 (M-003571) or nonspecific control growth were seeded at 1 Â 105 cells/ml of fresh RPMI 1640 siRNA (D-001206-13-05) obtained from Dharmacon Tech medium (Gibco BRL,Grand Island, NY, USA), supplemented (Lafayette, Co, USA) and electroporated in a 0.4-cm cuvette with 10% fetal calf serum, 2 mmol/l L-glutamine, penicillin G using the Gene Pulser electroporation apparatus (Bio-Rad (100 U/ml), streptomycin (100 mg/ml) (Gibco BRL) and with or Laboratories Inc., Herculex, CA, USA) and using a single-pulse without the compounds described above, in a humidified protocol (voltage 260 V and capacitance 1050 mF). At 48 h after mM atmosphere of 95% air/5% CO2. transfection, cells were treated with ATO 1 for another 48 h. The patients studied were affected by typical hypergranular The cells were harvested for DCm, annexin V, sub-G1 DNA APL (M3). Patients # 1 and # 2 were investigated at diagnosism content detection and immunoblotting. and patient # 3 at the time of relapse. Patient # 3 relapsed after 26 receiving ATRA þ idarubicin (AIDA regimen) as front-line Molecular analysis treatment. In all analyzed samples, the percentage of leukemic a infiltration exceeded 80%. The disease-specific PML/RAR Cell lysis, immunoblotting, immunoprecipitation, and ERK fusion transcript was detected in all cases by RT-PCR as 17,28,29 26 immunoenzymatic assay were carried out as described. described previously. For immunoprecipitation, the following antibodies were used: Peripheral blood was obtained after informed consent. rabbit polyclonal anti-p44/p42 ERK, rabbit polyclonal anti-Bad Leukemia cells were isolated and enriched on Ficoll–Hypaque and rabbit polyclonal anti-phospho–Bad, all provided by Cell density gradients (Pharmacia LKB Biotechnology AB, Uppsala, Signaling Technology Beverly, MA, USA.
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