Article Targeting iron homeostasis induces cellular differentiation and synergizes with differentiating agents in acute myeloid leukemia Celine Callens,1,2 Séverine Coulon,1,2 Jerome Naudin,1,2,3,4 Isabelle Radford-Weiss,2,5 Nicolas Boissel,4,9 Emmanuel Raffoux,4,9 Pamella Huey Mei Wang,3,4 Saurabh Agarwal,3,4 Houda Tamouza,3,4 Etienne Paubelle,1,2 Vahid Asnafi,1,2,6 Jean-Antoine Ribeil,1,2 Philippe Dessen,10 Danielle Canioni,2,7 Olivia Chandesris,2,8 Marie Therese Rubio,2,8 Carole Beaumont,4,11 Marc Benhamou,3,4 Hervé Dombret,4,9 Elizabeth Macintyre,1,2,6 Renato C. Monteiro,3,4 Ivan C. Moura,3,4 and Olivier Hermine1,2,8 1Centre National de la Recherche Scientifique UMR 8147, Paris 75015, France 2Faculté de Médecine, Université René Descartes Paris V, Institut Fédératif Necker, Paris 75015, France 3Institut National de la Santé et de la Recherche Médicale (INSERM), U699, Paris 75018, France 4Faculté de Médecine, Université Denis Diderot Paris VII, Paris 75018, France 5Laboratoire de cytogénétique, 6Laboratoire d’Hématologie, 7Service d’Anatomo-Pathologie, and 8Service d’Hématologie, Hôpital Necker-Enfants Malades, Assistance Publique Hôpitaux de Paris (AP-HP), Paris 75015, France 9Service d’Hématologie, Hôpital Saint-Louis, AP-HP, Paris 75010, France 10Unité de Génomique Fonctionnelle, Institut Gustave Roussy, Villejuif 94800, France 11INSERM U773, Centre de Recherche Biomédicale Bichat Beaujon CRB3, Paris 75018, France Differentiating agents have been proposed to overcome the impaired cellular differentia- tion in acute myeloid leukemia (AML). However, only the combinations of all-trans retinoic acid or arsenic trioxide with chemotherapy have been successful, and only in treating acute promyelocytic leukemia (also called AML3). We show that iron homeostasis is an effective CORRESPONDENCE target in the treatment of AML. Iron chelating therapy induces the differentiation of Olivier Hermine: leukemia blasts and normal bone marrow precursors into monocytes/macrophages in a [email protected] manner involving modulation of reactive oxygen species expression and the activation of OR Ivan Cruz Moura: mitogen-activated protein kinases (MAPKs). 30% of the genes most strongly induced by [email protected] iron deprivation are also targeted by vitamin D3 (VD), a well known differentiating agent. Iron chelating agents induce expression and phosphorylation of the VD receptor (VDR), and Abbreviations used: AML, acute The Journal of Experimental Medicine myeloid leukemia; APL, acute iron deprivation and VD act synergistically. VD magnifies activation of MAPK JNK and the promyelocytic leukemia; ATO, induction of VDR target genes. When used to treat one AML patient refractory to arsenic trioxide; ATRA, chemotherapy, the combination of iron-chelating agents and VD resulted in reversal of all-trans retinoic acid; CI, combination index; DFO, def­ pancytopenia and in blast differentiation. We propose that iron availability modulates eroxamine; DFX, deferasirox; myeloid cell commitment and that targeting this cellular differentiation pathway together DHE, dihydroethidium; with conventional differentiating agents provides new therapeutic modalities for AML. DHR123, dihydrorhodamine 123; FAB, French-American- British; LIP, labile iron pool; MAPK, mitogen-activated Acute myeloid leukemia (AML) is a hetero­ leading to AML leukemogenesis occur as a protein kinase; MDS, myelodys­ geneous malignant disorder originating from multistep process (Kelly and Gilliland, 2002; plastic syndrome; MFI, mean mutations in progenitor cells that lead to the Gilliland et al., 2004). Those events are broadly fluorescence intensity; MGG, unrestrained proliferation of undifferentiated classified into two groups: gene alterations that May Grünwald Giemsa; miRNA, micro RNA; mRNA, myeloblasts (Löwenberg et al., 1999). There is confer a proliferative and/or survival advan­ messenger RNA; NAC, a general consensus that the molecular events tage to hematopoietic progenitors (e.g., RAS, N-acetyl-l-cysteine; PDTC, pyrrolidine dithiocarbamate; PI, © 2010 Callens et al. This article is distributed under the terms of an Attribu- propidium iodide; ROS, tion–Noncommercial–Share Alike–No Mirror Sites license for the first six months reactive oxygen species; SCF, I.C. Moura and O. Hermine contributed equally to this paper. after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial– stem cell factor; VD, vitamin C. Callens’ present adress is Laboratoire d’Hématologie, Hôpital Share Alike 3.0 Unported license, as described at http://creativecommons.org/ D3; VDR, VD receptor. Necker-Enfants Malades, AP-HP, Paris, 75015 France. licenses/by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Exp. Med. Vol. 207 No. 4 731-750 731 www.jem.org/cgi/doi/10.1084/jem.20091488 Figure 1. Iron deprivation induces monocyte differentiation of AML cells. (a) Unsupervised microarray analysis of HL60 cells after a 48-h exposure to 10 µg/ml of anti-TfR1 antibody (mAb A24), 5 µM DFO, or 3 µM DFX. The color intensity represents the ratio of expression in drug-treated compared with control cells. The relative overexpression and underexpression compared with controls are shown in red and green, respectively (top). A Venn diagram of genes induced by iron deprivation (bottom) shows that only a subset of genes (105) have a shared altered expression between the 732 Iron chelation drives AML differentiation | Callens et al. Article FLT3, or KIT mutations) and gene alterations/point mu­ Like ATRA, VD belongs to the steroid superfamily and tations in transcription factors or transcriptional coactivators plays a critical role in regulating numerous cellular and physi­ (e.g., CEBP and NPM1) that affect differentiation (Fröhling ological responses. VD associates with its cognate receptor et al., 2005; Roumier et al., 2006; Renneville et al., 2008). (VDR) to form a heterodimer with the retinoid X receptor. The most important transcription factors that have been impli­ This complex then binds to the VD responsive element, cated in the myelomonocytic development are PU­1 for early which is present in the promoter region of target genes, myeloid commitment, C/EBP for granulocyte maturation, and thereby inducing changes in gene transcription (Hughes vitamin D3 (VD) receptor (VDR) and MafB for monocyte­ et al., 2009). VD also activates a variety of protein kinases, macrophage differentiation (Sieweke and Graf, 1998; Friedman, such as the three families of mitogen­activated protein kinase 2002; Mueller et al., 2002; Gemelli et al., 2008). (MAPK; ERK, JNK, and p38), protein kinase C, or Src fam­ The potential of the currently available cytotoxic chemo­ ily kinases by alternative signaling pathways which are not therapies to treat AML might have reached its limit (Löwenberg fully characterized (Wang et al., 2003). The active metabolite et al., 1999; Ravandi et al., 2007). Moreover, the majority of of VD, 1,25(OH)2D3, which is known as calcitriol, has anti­ patients with AML are 60 yr of age or older and, in this proliferative effects, can activate apoptotic pathways, and population, high­dose chemotherapy is associated with a can inhibit angiogenesis (Deeb et al., 2007). In the hemato­ high rate of morbidity and mortality. Although results of poietic system, numerous studies on AML blasts, leukemic new therapeutic strategies have improved steadily in younger cell lines, and primary myeloid precursors showed that VD adults over the last 20 yr, there have been fewer improve­ induces cell differentiation into monocytes (Abe et al., 1981; ments in outcome among elderly patients (Löwenberg et al., Koeffler et al., 1984; Hmama et al., 1999; Bastie et al., 2004; 2009). Based on the emerging knowledge of the biology of Ji and Studzinski, 2004; Gemelli et al., 2008; Hughes et al., AML, new targeted therapeutic approaches need to be devel­ 2009). Encouraging clinical trials have suggested that VD oped to interrupt proliferation, induce apoptosis, and/or could be effective as a differentiating agent in the treatment override the differentiation block. of AML and myelodysplastic syndrome (MDS), but hypercal­ The current treatment modality for the induction of a cemia has limited its use (Ferrero et al., 2004; Srivastava and terminal differentiation program in AML has only been Ambrus, 2004; Deeb et al., 2007). achieved in acute promyelocytic leukemia (APL or AML3) Iron (Fe) is required as a cofactor for several critical cellu­ by the use of differentiating agents combined with chemo­ lar enzymes involved in energy metabolism and cell prolifera­ therapy (Chen et al., 1994; Zhu et al., 2001; Wang and Chen, tion and is therefore essential for all living cells. Iron exists in 2008). In most of the cases, APLs are characterized by the two oxidation states, the ferrous (Fe2+) and the ferric (Fe3+) translocation t(15;17), which encodes a fusion of the promy­ forms, which participate to the generation of reactive oxygen elocytic leukemia and retinoic acid receptor proteins. The species (ROS) through the Fenton reaction. In this process, use of all­trans retinoic acid (ATRA) with chemotherapy was free cytosolic iron generates hydroxide anions and hydroxyl 2+ 3+ a major breakthrough in APL therapy. It has dramatically radicals from hydrogen peroxide (Fe + H202↔Fe + improved the prognosis from poor to a high cure rate (90% OH + OH•; Fenton et al., 1964) which can lead to cellular of patients; Wang and Chen, 2008). The use of arsenic triox­