radiation and chemotherapy [15], a hypoxic microenvironment may promote resistance of LSCs, suggesting that the hypoxia itself could be a therapeutic target. HIF-1a is a key transcriptional regulator of cellular response to hypoxia. HIF-1a subunits are normally degraded by the protein encoded by the von Hippel-Lindau tumor suppressor gene VHL (pVHL) in the presence of oxygen but are stabilized under conditions of hypoxia. Activation of the HIF-1a signaling pathway induces a vast array of gene products controlling energy metabolism, glycolysis, angiogenesis, apoptosis, and the cell cycle [16,17]. Recent genetic studies have shown that normal HSCs stabilize HIF-1a protein critical for maintaining cell cycle quiescence [18] and utilize glycolysis instead of mitochondrial oxidative phosphorylation [19]. HIF-1a is overexpressed in a broad range of human cancer types and increased levels of HIF-1a activity are often associated with increased tumor aggressiveness and therapeutic resistance [16]. HIF-1a may be clinically significant in acute leukemia as the protein has been shown to be overexpressed in clusters of leukemic cells in the BM of pediatric ALL patients [20]. Novel strategies to target HIF-1a in tumors are being developed to counter hypoxia’s pro-survival effect on cancer cells [21–23]. These strategies include inhibition of signal transduction pathways that have an impact on HIF-1a and direct inhibition of HIF-1a expression or activity by antisense or small molecules [21,24]. A disadvantage in focusing on HIF-1a as a chemotherapeutic target is that there are other HIF subunits (e.g HIF-2a) that are functional during hypoxia and the importance of the various subunits depends on which cell type is involved [25,26]. A second problem with targeting HIFs is that they are regulated in a complex manner by many factors other than oxygen concentra- tion, and can also be stabilized by mutation of VHL, so not all hypoxic cells express HIFs and conversely not all HIF-expressing cells are hypoxic. While HIFs may be valid targets in their own right, bioreductive prodrugs potentially provide a more direct mechanism for sensing and eliminating hypoxic cells. A more effective means of therapy would involve the use of an agent that is inert in normoxic conditions but is converted to a lethal form in the absence of oxygen. Such agents do exist and include tirapazamine and AQ4N [23,27]. Among the agents currently under investigation, PR-104 is one of the more promising candidates. PR-104 is a phosphate ester that is rapidly hydrolyzed in vivo to the corresponding alcohol PR-104A. Under extremely low oxygen concentrations, PR-104A is reduced mainly to the amine and hydroxyl-amine nitrogen mustards that induce DNA cross-linking in hypoxic cells [28,29]. In this study, we evaluated the role of hypoxia in the leukemic microenvironment, with particular attention to its relation with chemoresistance of leukemic cells. We show for the first time that HIF-1a is expressed in BM biopsies of ALL patients at diagnosis and that hypoxia areas are vastly expanded in leukemic BM, in which it contributes to microenvironment-mediated chemoresistance. Hyp- oxia-activated prodrug PR-104 demonstrated remarkable single agent activity in in vivo ALL models, indicating feasibility of exploiting hypoxia in the setting of acute leukemias. Altogether, these findings suggest that hypoxia plays a role in microenvironment-mediated chemoresistance in ALL and strongly suggests that hypoxia-targeting agents may be effective new tools in the treatment of ALL. Results Expansion of hypoxic niches within leukemic BM To examine the extent of hypoxia in leukemic vs. normal BM, we determined the levels of hypoxia in the BM microenvironment Targeting Leukemia-Associated Hypoxia with PR-104 of control NOD/Scid/IL2Rc-KO (NSG) mice and mice xeno- grafted with human or murine leukemic cells. To evaluate hypoxia, we utilized the specific chemical marker of hypoxia pimonidazole (PIM), a 2-nitroimidazole compound that under hypoxic conditions is metabolized and forms stable adducts inside the cells that can then be detected with a specific antibody. First, we validated specificity of PIM staining in ALL cells exposed to different oxygen levels. In leukemic cell lines REH and NALM6, PIM staining measured by flow cytometry was positive at 1% oxygen and further increased at 0.1% oxygen, indicating specific binding of this nitro-imidazole compound to hypoxic ALL cells (Fig. S1). We next utilized PIM immunohistochemistry (IHC) to assess levels of hypoxia in the BM of immunodeficient mice used as recipient for human leukemia engraftment. As shown in Figure 1A, PIM staining showed few discrete areas of positivity in control mice not injected with leukemic cells. In contrast, there was a great expansion of the hypoxic areas in the BM of the NSG mice injected with primary human B-ALL cells, with a uniform pattern of PIM positivity that co-localized with CD22+ staining of ALL cells (Figure 1B). To examine the role of hypoxia using immune competent mice, we made use of a syngenic model of blastic phase chronic myelogenous leukemia (CML) whereby C57Bl6/J mice were injected with murine bone marrow cells expressing the BCR- ABL and Nup98 translocation products, and green fluorescent protein GFP [30]. In this model, the progression of leukemia (by GFP staining) and development of hypoxia (using PIM as above) in the BM and spleen was followed over time (Figure 2). Twenty hours after injection of the cells, there were very few cells showing positive signal in both tissues analyzed. Three days after cell injection GFP positivity became apparent, with several areas of PIM positivity. By day 6 after injection, both BM and spleen exhibited vast areas of PIM positivity (Figure 2). This rapid progression of leukemia was accompanied by notable changes in the BM vasculature revealed by lectin labeling of blood vessels (Fig. S2). Modest decrease in the microvessel density was observed in BM from leukemia-bearing mice compared to healthy controls. In addition, the vasculature in the leukemic BM appeared morphologically different than that in the normal BM, with elongated and distorted vessels. These findings indicate that expansion of hypoxia in the in vivo murine models of leukemia is accompanied by a loss of vessel integrity with increased leukemic cell burden. To validate these findings in human BM specimens, we evaluated expression of HIF-1a, as an established marker of hypoxia, by quantitative immunohistochemistry using CRi Nuance spectral imaging system. BM biopsy specimens from 9 ALL patients at the time of diagnosis and after induction chemotherapy upon recovery of normal hematopoiesis, were stained with HIF-1a antibody (Figure 3). As controls, HIF-1a immunostaining was assessed in BM sections from 3 normal donors. HIF-1a was expressed at very low levels in the normal bone marrow (Figure S3A). In agreement with our observations in mice, HIF-1a was strongly positive in 6 of the 9 BM biopsies obtained from ALL patients at the time of diagnosis (see examples in Figure 3). In contrast, HIF-1a signal was expressed in only a few cells in the paired BM samples obtained after patients have achieved complete remission (CR). Importantly, HIF-1a was expressed not only in leukemic blasts, but also in the surrounding stromal cells (Figure S3B), indicating expansion of the hypoxic niches. In 3 out of the 6 patients the levels of HIF-1a at CR were very close to those observed in normal BMs (indicated by the continuous line on Fig. 3B). These results suggest stabilization of HIF-1a protein in BM infiltrated with leukemic cells and possible PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e23108