Targeting Leukemia-Associated Hypoxia with PR-104 Figure 5. In vitro hypoxia cytotoxicity of PR-104A. (A) B-cell ALL cell lines Nalm6 and REH B-ALL were treated with PR-104A under normoxic or hypoxic conditions for 6 hours. Cell death, measured as percentage of Annexin V (AnnV)–positive cells and cell number were measured 48 hours later by FACS. (B) and (C) Three primary ALL samples (B) or three control CB (C) were treated with PR-104A at normoxic or hypoxic conditions for 6 hours. Cell death, measured as percentage of Specific Apoptosis [% Specific Apoptosis = (%AnnV sample-%AnnV control)/(100-%AnnV control)*100] were measured 24 hours later by FACS. *P,0.05; **P,0.01; ***P,0.0001. doi:10.1371/journal.pone.0023108.g005 physiological conditions the BM harbors only discrete areas of hypoxia. Our exploration of the role of hypoxia in leukemic cells showed that, in vitro, 1% O2 levels conferred resistance against selective chemotherapeutic agents in the ALL cell lines tested. The mechanism responsible for this effect was not addressed in the present work. However, a potential candidate is HIF-1a given its role as master regulator of the hypoxic response. Many known HIF-1a targets could mediate the protection conferred by hypoxia, and a number of those have been validated as chemoresistance factors in leukemias (i.e., MDR-1, Nur-77, CXCR4 and others) [16,42]. In this regard, we have previously reported that hypoxia increases CXCR4 expression leading to increased migration and survival of leukemic cells [43]. HIF-2a is another HIF family member induced by chronic hypoxia and expressed at high levels in primary tumors and their metastases [25,44]. HIF-2a targets overlap with but seem to be different from those regulated by HIF-1a [26,45,46]. Which HIF-1a targets mediate hypoxia-induced chemoprotection in leukemia cells and whether there is any contribution from HIF-2a requires further investigation. In preliminary studies, HIF-2a was not found to be expressed in the ALL cell lines used for this study. Furthermore, HIF1a-independent mechanisms have been described as mediat- ing hypoxia responses in various systems. Among them, NF-kB is of particular interest given that it can be induced by hypoxia and can regulate important pathways including cell proliferation, angiogenesis, metastasis and survival [47]. Its importance in hematological malignancies is becoming clearer [48,49] and in fact, two groups [50,51] recently demonstrated that modulation of the NF-kB pathway sensitized leukemic cells to chemotherapy and inhibited leukemia cells growth, respectively. While hypoxia is a known factor mediating chemoresistance in solid tumors, our data presented here for the first time indicate the relevance of these processes in leukemia pathophysiology. Various approaches have been explored to target the hypoxic microenvironment and thus render tumor cells susceptible to chemotherapy. Some approaches seek to directly inhibit HIF-1a activity, while others take advantage of the hypoxic microenvi- ronment via hypoxia-activated prodrugs. In this study we used PR- 104, a nitrogen mustard that under very low oxygen concentra- tions is reduced to its amine and hydroxyl amine metabolites which function as alkylating agents, leading to cell death. In vitro, the alcohol form of PR-104 had good cytotoxic activity against B- lineage ALL cell lines REH and Nalm6 and primary ALL cells. As expected, the cell killing effect was more pronounced in hypoxic compared to normoxic conditions. In cord blood cells used here as normal controls, the drug caused little cell death. The oxygen concentration required for 50% inhibition of PR-104A cytotox- PLoS ONE | www.plosone.org 7 August 2011 | Volume 6 | Issue 8 | e23108