Targeting Leukemia-Associated Hypoxia with PR-104 Figure 7. PR-104 induced decrease of circulating CD45+ cells and of hypoxia in NSG mice injected with a primary B-lineage ALL sample. (A) Time course of changes in percentage of circulating CD45+ cells determined in peripheral blood by FACS (n = 9/group). Arrows at the bottom indicate days when PR-104 was administered. (B) PIM in BM flushes from two control and three PR-104-treated mice determined by FACS. PIM was administered 3 hour prior to sacrifice on Day 52. PIM MFI is plotted as a ratio between the sample MFI/unstained control MFI. Bottom panel: percentage of PIM positive cells. The % of human CD45 positive cells in peripheral blood (PB) is indicated for each mouse. (C) Pimonidazole adducts and H&E staining in BM sections from control and PR-104-treated mice determined by IHC. Original magnification is indicated. doi:10.1371/journal.pone.0023108.g007 xenograft which showed the highest sensitivity to PR-104, to very low levels of expression. Therefore, one could speculate that it might relate to: (1) the known sensitivity of ALL to alkylating agents e.g. cyclophosphamide; (2) the homologous recombination repair status of the cells; (3) the difference in models tested (systemic model of leukemia versus subcutaneous solid tumors) and differences in PR- 104 biodistribution that might impinge on this; (4) other unknown factors intrinsic to the different tumor types. Notably, another hypoxia-activated alkylating agent TH-302 exhibited remarkable anti-tumor effects on multiple disease parameters in a multiple myeloma model [40]. In all three models tested here, restoration of normal hematopoiesis resulted in drastic decline in the areas of PIM positivity possibly indicating normalization of BM vasculature and restoration of higher physiologic oxygen tension. It is conceivable to hypothesize that application of hypoxia-activated prodrugs would selectively eliminate leukemic cells in most hypoxic areas. We also propose that leukemic stem cells may have a propensity similar to HSC to reside in these areas resulting in the emergence of resistant clones. While these hypotheses require further experimental proof, they may develop into rational combinations of PR-104 with chemotherapeutic or other targeted agents. Another attractive approach would be the utilization of this class of drugs in the setting of minimal residual disease. However, given the recent finding that normal long-term repopulat- ing cells may likewise reside in these areas, their elimination may result in prolonged myelosuppression. Indeed, myelosupression has been reported for PR-104 in a Phase I clinical trial in solid tumors [31]. It is arguable however whether this side effect is attributable to the ability of PR104 to target human progenitor cells residing in hypoxic areas, or to the fact that CD34 positive progenitors express AKR1C3, the enzyme responsible for aerobic activation of PR-104 [35]. In this respect, PR104-A indeed caused significant inhibition of the clonogenic survival of human progenitor cells in vitro, which was in turn ameliorated by AKR1C3 inhibitor naproxen (personal communications, J. Down and K. Parmar). Further studies with more selective hypoxia-activated prodrugs or HIF inhibitors will likely yield the answer to this clinically relevant question. In summary, we have shown for the first time that the leukemic BM microenvironment is hypoxic and provided a rationale for targeting it. Our results with the bioreductive drug PR-104 suggest that targeting hypoxia is feasible and could have significant impact in the treatment of leukemias. Several in vivo murine leukemia models validated hypoxia as a potential target; as a consequence, PR-104 is undergoing a Phase I clinical trial in patients with relapsed and refractory AML and ALL. In the future, the ultimate goal would be to use bioreductive drugs in combination with conventional chemotherapies or novel targeted agents to eradicate resistant leukemic blasts that persist in the hypoxic BM microenvironment. Materials and Methods Chemicals, antibodies, and reagents PR-104 and PR-104A were provided by Proacta. Anti-human AKR1C3 and anti-b-actin mAb were purchased from Sigma- PLoS ONE | www.plosone.org 9 August 2011 | Volume 6 | Issue 8 | e23108