Targeting Leukemia-Associated Hypoxia with PR-104 Figure 1. Expansion of the hypoxic BM niche in an ALL xenograft model. Representative images from one healthy control mouse (two different BM fields, A) or mice transplanted with CD22-expressing leukemic cells from an infant with MLL-rearranged B-lineage ALL (B). After 52 days, mice were injected with pimonidazole (PIM) 3 hrs prior to sacrifice. Areas of hypoxia were detected by PIM antibody, and leukemic cells by anti-CD22. Original magnification is shown next to each panel. doi:10.1371/journal.pone.0023108.g001 reinstitution of higher oxygen levels upon restoration of normal hematopoiesis. To determine whether hypoxia influenced the sensitivity of leukemic cells to chemotherapy, in vitro experiments were performed to compare the effects of standard chemotherapeutic agents at different oxygen levels. REH and Nalm6 B-lineage ALL cells were exposed to various chemotherapy agents (vincristine, methotrexate, doxorubicin, or etoposide) for 48 hours under either normoxic (21% O2) or hypoxic (1% O2) conditions. Effects on viability and cell death were determined by flow cytometry. At 21% O2, all drugs induced cell killing in a dose-dependent manner. By contrast, a significant decrease in growth inhibition (the hypoxic drug values were normalized to the hypoxic control) and apoptosis induction was noted in REH and NALM6 cells treated with vincristine, methotrexate, and etoposide under hypoxic conditions (Figure 4A). A minimal effect was observed for doxorubicin, however not as impressive as the hypoxia induced-chemoprotection against the other drugs tested. In vitro hypoxia-selective cytotoxicity of PR-104 Given the evidence that hypoxia mediates chemoresistance of leukemic cells, we sought to exploit the hypoxic microenvironment of leukemia by utilizing hypoxia-activated pro-drugs. We first determined the hypoxia selectivity of DNA-cross linking agent PR- 104 against leukemia cell lines. To examine PR-104 cytotoxic activity, cells were treated with the alcohol form of the drug (PR- 104A) for 6 hours at 21% or 1% O2, and cell death was assessed by FACS determination of Annexin V staining at 48 hours. Concentrations were selected based on the therapeutic levels achievable in humans, which provides a plasma AUC of ,40 mM- h at the MTD of 1100 mg/m2 in patients with solid tumors [31]. In REH and Nalm6 pre-B ALL cells, PR-104A induced greater growth inhibition at 1% O2 compared to 21% O2 culture conditions (Figure 5A). While PR-104A toxic effect was maximal under hypoxia, there was some degree of drug-induced cell death in both ALL cell lines even in normoxic conditions. Even though activation of PR-104 requires very low oxygen levels, Guise et al. reported that PR-104 can also be activated by the aldo keto reductase enzyme prostaglandin F synthase, 17b-hydroxysteroid dehydrogenase type V (AKR1C3) independent of oxygen level [32]. Since we observed cytotoxicity, although diminished, at ambient oxygen levels, we evaluated the presence of the AKR1C3 enzyme in the three cell lines tested. As shown in the Western blot in Figure S4, when compared to the SKOV3 cells used as positive control, Nalm6 did not express detectable levels of the enzyme, while REH cells did. This observation is consistent with the fact that REH exhibited higher sensitivity to PR-104A in normoxia than Nalm6 cells. PR-104A activity was next tested in primary ALL samples. Figure 5B shows that PR-104A also had higher activity against primary leukemia cells at 1% compared to 21%O2. However, in primary samples PR-104A induced apoptosis, albeit at less extent, at 21% O2, which led us to check the levels of AKR1C3 in samples from patients as well as normal donors. We found that patient samples have in general higher levels of AKR1C3 than CD34+ cells isolated from normal donors, in agreement with high AKR1C3 message levels reported by Birtwistle et. al. for AML patients [33] (Fig S4B and C). AKR1C3 levels were variable, but one third of the ALL samples showed high levels suggesting that high AKR1C3, in addition to hypoxia, might also be a target in some leukemias. This observation could explain the PR-104A sensitivity observed at 21% O2 and also provides an additional rationale for the use of PR-104, since this drug would be activated not only under hypoxic conditions but also selectively by leukemia cells expressing high levels of the enzyme. We also tested the activity of PR-104A against mononuclear cells isolated from cord blood samples, used here as a normal control (Figure 5C). PR- 104A did not induce apoptosis in these cells either at 21% or at 1 %O2 (N = 3). In vivo PR-104 antitumor activity To test the in vivo efficacy of PR-104, several leukemia models were employed. Our first xenograft model focused on using Nalm6 cells transduced with Luciferase. NSG mice injected with Nalm6- GFP/Luciferase cells were treated with PR-104 starting on day 3 after injection (250 mg/kg, i.p. 3 times a week for 2 weeks), and PLoS ONE | www.plosone.org 3 August 2011 | Volume 6 | Issue 8 | e23108