Leukemia (2013) 27, 2220–2228 & 2013 Macmillan Publishers Limited All rights reserved 0887-6924/13 www.nature.com/leu ORIGINAL ARTICLE The novel arsenical Darinaparsin circumvents BRG1-dependent, HO-1-mediated cytoprotection in leukemic cells N Garnier1,2, LA Petruccelli1,2, MF Molina1,2, M Kourelis1,2, S Kwan1,2, Z Diaz1,2, HM Schipper2, A Gupta3, SV del Rincon1,2, KK Mann1,2 and WH Miller Jr1,2 Darinaparsin (Dar) is a more potent cytotoxic arsenical than arsenic trioxide (ATO). We hypothesized that the increased cytotoxicity of Dar may be because of a decreased cytoprotective response. We observed that, unlike ATO, Dar does not induce heme oxygenase-1 (HO-1), even though it induces expression of other nuclear factor (erythroid-derived 2)-like 2 (NRF2)-dependent detoxifying enzymes to a greater extent than ATO, in both cancer cell lines and patient-derived leukemic cells. This strengthens the emerging evidence, showing that response to reactive oxygen species (ROS) is stimuli specific. Dar treatment prevents recruitment of the transcriptional coregulator Brahma-related gene 1 (BRG1) to the HMOX1 promoter, which is required for HMOX1 expression. The inability of Dar to induce HO-1 correlates with arrest in G2/M cell cycle phase and BRG1 phosphorylation. Inhibition of HO-1 increases the toxicity of ATO, but has no effect on Dar-induced apoptosis. Accordingly, the lack of HO-1 induction is involved in Dar’s enhanced antileukemic properties. Our data highlight cytoprotective responses mediated by HO-1 and BRG1 as a novel target for enhancing the therapeutic range of arsenicals. Leukemia (2013) 27, 2220–2228; doi:10.1038/leu.2013.54 Keywords: arsenic; leukemia; Darinaparsin; HO-1; BRG1; NRF2 INTRODUCTION we reviewed.1 We postulate that within the signaling differences Darinaparsin (Dar; ZIO-101, S-dimethylarsino-glutathione) is a lies the mechanism responsible for the enhanced antitumor effect promising new arsenic-based, anticancer drug candidate, of Dar. which is currently undergoing clinical studies in both hematolo- In response to oxidative stress, cells induce an arsenal of gical malignancies and solid tumors.1 This organic arsenical, protective proteins including the nuclear factor (erythroid-derived which consists of dimethylarsenic conjugated to glutathione, 2)-like 2 (NRF2) gene battery, which includes heme oxygenase-1 shows significant activity against multiple cancers in vitro.2 (HO-1, HMOX1 gene), NAD(P)H:quinone oxidoreductase (NQO1), Another arsenical, arsenic trioxide (ATO), is a proven thioredoxin-reductase-1 (TR1, TXNRD1 gene) and glutamate- 15 chemotherapy for acute promyelocytic leukemia (APL), but has cysteine ligase regulatory subunit (gGCS). NRF2 is a basic substantially reduced antitumor effects at clinically achievable leucine zipper transcription factor that mediates the expression of doses in other malignancies.3–5 We have shown previously that these key protective enzymes by binding antioxidant response Dar is a more potent inducer of apoptosis than ATO in various elements (ARE)16–18 in the regulatory regions of these genes. malignant cell lines and is highly active against APL cells.2 Under unstressed conditions, Kelch-like ECH-associated protein 1 Importantly, Dar has a maximum tolerated dose that is 50-fold confines NRF2 in the cytoplasm, where it is constitutively higher than ATO in mice and is active against xenograft tumors.6 degraded.19–21 In contrast, ROS liberate NRF2 from Kelch-like However, the mechanisms of its increased antitumor efficacy have ECH-associated protein 1, leading to NRF2 translocation and not been elucidated. accumulation in the nucleus.15 Transcriptional regulation of some Oxidative damage is postulated to be a key mechanism by of these detoxifying enzymes involves a competition between the which arsenicals initiate the apoptotic process.7–12 Consistent with activator NRF2 and the repressor BTB, and CNC homolog 1 its increased cytotoxicity, we have reported that Dar induces (BACH1) for interaction with their AREs.22 Of the NRF2 target - significantly more superoxide (O2 ), hydrogen peroxide and genes, regulation of HMOX1 expression is unique because it is oxidative damage than ATO in arsenic-sensitive NB4 and further regulated by the binding of Brahma-related gene 1 (BRG1, arsenic-resistant AR2 cells.2 Both ATO and Dar-induced ROS SMARCA4 gene), the catalytic subunit of SWI2/SNF2-like chromatin- 23 activate the c-Jun NH2-terminal kinase/stress-activated protein remodeling complex, to the ARE. kinase pathway, leading to apoptosis.2,13,14 However, we have also Here, we show that Dar selectively fails to induce HO-1 reported that Dar triggers apoptosis by inducing cellular expression in leukemic cell lines in vitro and in APL patient blasts responses that do not completely overlap with ATO.1,2 The ex vivo, even though it induces the expression of other NRF2 characterization of similarities versus differences in triggered target genes. We provide evidence that Dar prevents BRG1 pathways and intracellular ROS modulation between ATO and recruitment to the HMOX1 promoter. We also show that specific Dar have elicited interest among the labs working on arsenic, as inhibition of HO-1 enhances the toxicity of ATO, but not Dar, in 1Department of Oncology, Lady Davis Institute for Medical Research, Segal Cancer Center, Sir Mortimer B. Davis Jewish General Hospital, McGill University, Montre´al, Quebec, Canada; 2Faculty of Medicine, Department of Experimental Medicine, McGill University, Montre´al, Quebec, Canada and 3Osta Biotechnologies, Inc., Dollard-des-Ormeaux, Quebec, Canada. Correspondence: Dr WH Miller Jr, Department of Oncology, Lady Davis Institute for Medical Research, Segal Cancer Centre, Sir Mortimer B. Davis Jewish General Hospital, McGill University, 3755 Coˆte Ste-Catherine Road, Montreal, Quebec, Canada H3T 1E2. E-mail: [email protected] Received 9 November 2012; revised 4 February 2013; accepted 11 February 2013; accepted article preview online 21 February 2013; advance online publication, 15 March 2013 Darinaparsin prevents heme oxygenase-1 induction N Garnier et al 2221 leukemic cells. This suggests that the inability of Dar to induce have previously shown that Dar induces more ROS than ATO.2 HO-1 expression is partially responsible for the increased We hypothesized that the enhanced cytotoxicity of Dar is because pro-apoptotic potential we observe. of its inability to elicit a complete antioxidant enzyme response. Therefore, we investigated whether Dar induced significant levels of HO-1, the prototypic arsenic-induced phase II antioxidant MATERIALS AND METHODS enzyme. Figure 1b shows that Dar did not induce HO-1 protein Ethics statement expression in any of the tested cell lines, as opposed to ATO that The patient samples were obtained, collected and used in accordance to strongly induced expression in all cell lines. We were able to the ethical requirements and regulations of the Jewish General Hospital. reproduce these results in HeLa cells, suggesting that our results Informed written consent was obtained from all patients involved. are not limited to hematological malignancies (Figures 1a and b). To verify this observation, we treated both NB4 and U937 cells Cell culture with ATO or Dar and assessed HMOX1 mRNA levels by quantitative All cells were grown in a humidified chamber at 37 1C with 5% CO2. NB4, PCR (qPCR). Treatment with Dar failed to induce significant HMOX1 U937, U266, Kasumi-1, LY8 and HeLa cells were obtained from the expression in either cell line. This result remained consistent in american type culture collection (ATCC) and cultured as recommended. NB4 (Figure 1c) and U937 (Figure 1d), regardless of treatment dose Blasts from APL patients were isolated from either peripheral blood or and/or duration. In addition, we tested all the cell lines in Figure 1a bone marrow (see Supplementary Materials). and found that Dar did not induce significant HMOX1 mRNA expression in any of them (Supplementary Figure 1). Cell viability and caspase-3/7 assay Cell viability and caspase-3/7 activity were assayed using CellTiter-Glo Dar fails to induce HO-1, but not other NRF2 target genes, in NB4 Luminescent Cell Viability Assay and Caspase-Glo 3/7 Assay (Promega, cells Madison, WI, USA) according to manufacturer’s instruction (see Supplementary Materials). We investigated whether Dar would induce significant levels of other phase II antioxidant enzymes, which are induced through activation of the NRF2-signaling pathway.15,17,18 NB4 cells were Propidium iodide staining treated for 3 or 6 h with increasing concentrations of ATO or Dar. As 2 Quantification of apoptotic cells was performed as previously described expected, HO-1 protein levels increased with ATO, but not with Dar (see Supplementary Materials). (Figure 2a). In contrast, Figures 2b, c and d show that both ATO and Dar increase, in a time- and dose-dependent manner, protein levels Protein quantification of TR1, gGCS and NQO1, with Dar consistently inducing expression Cells were lysed, whole-cell extracts were run on SDS-polyacrylamide gel at lower concentrations. To expand our observations, we also tested electrophoresis followed by western blot analysis (see Supplementary the ability of ATO and Dar to induce TR1 and gGCS protein levels in Table 1). Levels of phospho-DNA-PKcs (T2604) were measured by flow Kasumi-1 and U937, which are two non-APL cell lines. As expected, cytometry (see Supplementary Materials). NRF2 protein levels were both ATO and Dar increased protein levels of TR1 and gGCS assessed by densitometry using Image J (imagej.nih.gov). The intensity of each NRF2 band is normalized with the corresponding loading control (Supplementary Figure 2A and 2B). NQO1 protein expression is band (GAPDH or H3) and compared with the corresponding nuclear or shown as a time course because of the transient nature of its cytoplasmic control-treated bands, which were given the value of 1. expression. NQO1 protein levels increase quickly following exposure to arsenicals but decrease at 6 h (Supplementary Figure 2C). Our mRNA analysis data indicate that the activation of HO-1 expression in response to NRF2 signaling is different than for the other NRF2 target genes.