Targeting Leukemia-Associated Hypoxia with PR-104 Figure 6. PR-104 administration resulted in decreased tumor burden and hypoxic areas in a Nalm6/GFP ALL model. 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). PIM was administered 3 hours prior to sacrifice. (A) Luciferase activity in PR-104-treated and control mice over the course of the experiment. Bottom graph: Luciferase quantification of control and PR-104 treated mice. (B) BM sections from control and treated mice stained for the hypoxia markers PIM and CAIX and for GFP, CXCR4, CD31 and SDF1a. Original magnification, 6100 and 6400 (H&E) 640 and 6400 (PIM/GFP, CAIX, CXCR4, CD31 and SDF1a). doi:10.1371/journal.pone.0023108.g006 icity in SiHa cells is only 0.1 mM, corresponding to 0.01% oxygen in the gas phase [52]. The true oxygen concentration in cells with 1% oxygen in the gas phase is unknown, but is probably not low enough to fully activate PR-104A. However, given that PIM and PR-104A are both nitro compounds, and probably have broadly similar oxygen dependencies, we tested PIM activation in Nalm6 and REH under decreasing oxygen concentrations and observed hypoxia dependent binding at 1%O2 and lower (Figure S1). These findings suggest that PR-104 could similarly, although not fully, be activated at 1% oxygen and that therefore the in vitro activity probably underestimates activity in severely hypoxic tissues. Our results in vitro prompted us to test PR-104 in several in vivo leukemia models, where it showed remarkable antitumor activity as a single agent. Murine xenograft models harboring Nalm6- Luciferase ALL or primary ALL treated with PR-104 showed responses with significant decreases in the percentage of circulating CD45+ cells and prolongation of survival. Furthermore, the agent is likely effective against a broad range of acute leukemias as PR-104 was effective in reducing tumor burden when primary AML cells were used in the murine xenograft model. There were however differences in the overall responses to the treatment since mice injected with the aggressive MLL-ALL primary cells exhibited a gradual increase in circulating leukemia cells once the treatment was stopped, an event that was not observed in mice injected with primary AML. Dose response curves were also evaluated in two xenograft models of primary ALL. PR-104 significantly delayed the progression of ALL-8 in a dose-dependent manner at all doses tested. Treatment at the three highest doses of this drug resulted in an objective response measure (ORM) of maintained complete response (MCR). Two of these three doses correspond to the plasma levels of the drug that can be achieved in humans; recent comparison of plasma pharmacoki- netics has shown that the mouse dose equivalent to the MTD in humans with solid tumors (1100 mg/m2) is ,125 mg/kg [31]. PR- 104 also significantly delayed the progression of ALL-19 in a dose- dependent manner at the three highest doses tested. However, only the highest dose, which leads to plasma drug concentrations well above those achievable in the clinic, resulted in an ORM of MCR suggesting that combination therapies are needed. Since both ALL-8 and ALL-19 are derived from patients who experienced early relapse (within 2 years of diagnosis), possible differences in leukemia subtype specificity of PR-104 between T-lineage ALL (ALL-8) and B-cell- precursor ALL (ALL-19) should be explored in future studies. Interestingly, given the remarkable antitumor activity of PR-104, even at concentrations 50% or lower than the MTD, there seems to be higher activity of the agent in the leukemia models than that observed in the pediatric [35,53] or adult [28] solid tumours. The reasons for the observed differences are not clear at this point and would not appear to simply relate to AKR1C3 expression levels, since the levels in the 6 solid tumors varied from as high as the T-ALL PLoS ONE | www.plosone.org 8 August 2011 | Volume 6 | Issue 8 | e23108