Targeting Leukemia-Associated Hypoxia with PR-104 Figure 3. Expansion of the hypoxic BM niche in primary leukemia samples reverted following complete remission (CR). HIF-1a was detected by IHC in BM biopsies from 9 ALL patients at diagnosis and at CR after induction chemotherapy. (A) Representative images from 3 patients are shown. Original magnification, 6500. (B) Quantification of HIF-1a expression using Cri Inform software. A threshold was established based on the scores of three normal BM samples (continuous line represents average HIF-1a and dashed line correspond to SD for these 3 samples, respectively) CR; complete remission *** P,0.0001, error bars SEM. doi:10.1371/journal.pone.0023108.g003 sacrificed, and cells containing PIM adducts and therefore hypoxic were determined in BM flushes by flow cytometry. In control mice (n = 2), PIM intensity ratios in the BM flushes (normalized to the intensity for the same cells but without PIM antibody) were 20 and 10, respectively, and the percentage of PIM positive cells was 79 and 93%, respectively. In contrast, in the treated mice (n = 3), PIM intensity ratios decreased to 4 and 5, and the percentage of PIM positive cells was 20% and 40%, respectively, indicating that the level of hypoxia in the BM of these mice was significantly lower than in controls (Figure 7B). The extent of hypoxia in control vs. treated mice was additionally confirmed in BM sections from both groups by histochemical detection of PIM adducts after treatment (Figure 7C). In control mice, the BM was fully PIM positive, which correlated with the higher ratio of PIM intensity detected in BM flushes by flow cytometry. In treated mice, on the other hand, the BM had hypoxia-free zones (blue staining in Figure 7C) in accordance with the lower ratios of PIM intensity detected in BM flushes from these mice and also with the decreased percentage of circulating CD45+ cells. Next, anti-leukemia effects of PR-104 were evaluated in NOD/ Scid mice injected with cells from two pediatric ALL patients propagated in vivo (referred to as ‘‘xenografts’’, ALL-8 and ALL-19 [34]). In these studies, PR-104 was administered to several groups of mice in a dose-dependent manner over clinically relevant doses (200, 100 and 50 mg/kg). The maximum tolerated dose (MTD) of 550 mg/kg dose was also included for comparison. Compared to vehicle control, PR-104 significantly delayed progression of ALL-8 at all doses tested, resulting in significantly increased event-free survival (EFS) of mice in all treatment groups (Figure 8A, Table S1). The efficacy of PR-104 was dose dependent. The estimated leukemia growth delay (LGD) values, calculated according to established methodology [34,35], ranged from 100.9 to 33.5 days for the highest and the lowest dose of PR-104, respectively. In ALL-8 xenografted mice, the three highest doses of PR-104 produced objective responses, all of which were maintained complete responses (MCR), whereas treatment with the lowest dose resulted in stable disease (Figure 8B). Compared to vehicle control, PR-104 significantly delayed leukemia progression of ALL-19 at three of four doses tested, resulting in significantly increased EFS of mice in these treatment groups (Figure 8C, Table S1). Estimated LGD values ranged from .54.6 to 3.4 days for the highest and the lowest dose of PR-104, respectively. Only treatment at the highest dose of the drug resulted in the objective response of MCR (Figure 8D). Finally, the anti-leukemia effects of PR-104 were tested in against a primary xenograft using human AML cells. PR-104 at 250 mg/kg administered on day 72 after injection of NSG mice with primary refractory AML cells resulted in a statistically significant prolongation of survival when compared to control mice that did not receive therapy (median survival time (MST) of control and PR-104 treated mice 86 and 169 days, respectively, n = 5 mice/group; P = 0.026, not shown). During the course of the treatment, leukemia was assessed as circulating human CD45 positive cells (Figure S5A): while there was persistence or gradual increase in the percentage of circulating human CD45 positive cells in the PBS-treated mice, this population remained undetect- able in PR-104-treated mice. After treatment, leukemia engraft- ment was evaluated in different tissues by staining for CD45+. Whereas in control mice CD45+ cells had infiltrated lung, liver, bone, and spleen, in treated mice these cells were almost PLoS ONE | www.plosone.org 5 August 2011 | Volume 6 | Issue 8 | e23108