Pronounced Hypoxia in Models of Murine and Human Leukemia: High Efficacy of Hypoxia-Activated Prodrug PR-104 Juliana Benito1, Yuexi Shi1, Barbara Szymanska5, Hernan Carol5, Ingrid Boehm5, Hongbo Lu1, Sergej Konoplev3, Wendy Fang 4 , Patrick A. Zweidler-McKay4, Dario Campana6, Gautam Borthakur1, Carlos Bueso-Ramos3, Elizabeth Shpall2, Deborah A. Thomas1, Craig T. Jordan 7 , Hagop Kantarjian 1 , William R. Wilson 8 , Richard Lock8, Michael Andreeff1,2, Marina Konopleva1,2* 1 Section of Molecular Hematology and Therapy, Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America, 2 Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America, 3 Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America, 4 Department of Pediatrics, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America, 5 C25 Lowy Cancer Research Centre, Children’s Cancer Institute Australia for Medical Research, University of New South Wales, Randwick, New South Wales, Australia, 6 St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America, 7 School of Medicine and Dentistry, University of Rochester, Rochester, New York, United States of America, 8 Auckland Cancer Society Research Center, The University of Auckland, Auckland, New Zealand Abstract Recent studies indicate that interactions between leukemia cells and the bone marrow (BM) microenvironment promote leukemia cell survival and confer resistance to anti-leukemic drugs. There is evidence that BM microenvironment contains hypoxic areas that confer survival advantage to hematopoietic cells. In the present study we investigated whether hypoxia in leukemic BM contributes to the protective role of the BM microenvironment. We observed a marked expansion of hypoxic BM areas in immunodeficient mice engrafted with acute lymphoblastic leukemia (ALL) cells. Consistent with this finding, we found that hypoxia promotes chemoresistance in various ALL derived cell lines. These findings suggest to employ hypoxia-activated prodrugs to eliminate leukemia cells within hypoxic niches. Using several xenograft models, we demonstrated that administration of the hypoxia-activated dinitrobenzamide mustard, PR-104 prolonged survival and decreased leukemia burden of immune-deficient mice injected with primary acute lymphoblastic leukemia cells. Together, these findings strongly suggest that targeting hypoxia in leukemic BM is feasible and may significantly improve leukemia therapy. Citation: Benito J, Shi Y, Szymanska B, Carol H, Boehm I, et al. (2011) Pronounced Hypoxia in Models of Murine and Human Leukemia: High Efficacy of Hypoxia- Activated Prodrug PR-104. PLoS ONE 6(8): e23108. doi:10.1371/journal.pone.0023108 Editor: James McCubrey, East Carolina University, United States of America Received March 14, 2011; Accepted July 12, 2011; Published August 11, 2011 Copyright: 2011 Benito et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Cancer Institute, NOI-CM-42216 and NOI-CM-91001-03, P01 CA55164 (to MA), 1R21CA153019-01 and 1R01CA155056-01 (MK), UTMDACC Institutional Research Grant (to MK), the Paul and Mary Haas Chair in Genetics (to MA) and National Institutes of Health Cancer Center Support Grant CA016672 (to Flow Cytometry and Cellular Imaging Facility and Research Animal Support Facility at UT MD Anderson Cancer Center). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have read the journal’s policy and have the following conflicts: Dr. William R. Wilson holds stock in PROACTA Inc. and is a consultant to the company. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: mkonople@mdanderson.org Introduction Self-renewal, differentiation, and mobilization of normal human stem cells (HSCs) are the result of interactions between osteoblastic and vascular microenvironmental domains within the BM [1–6]. The osteoblastic niche comprises mainly osteoblasts on the internal surface of the bone and is thought to home quiescent, long-term HSCs. The vascular domain is formed by sinusoidal endothelial cells and is rich in actively proliferating short-term HSCs [7–9]. While the importance of the different microenviron- mental domains for HSC maintenance is still under investigation, there is evidence for a gradient of decreasing oxygen levels from the vascular to the osteoblastic niche and for the notion that the most primitive HSC are sequestered in a hypoxic microenviron- ment, implying that low oxygen levels play a fundamental role in the maintenance of normal stem cell function [10,11]. Quiescent BM cells from regions where blood perfusion is minimal (as determined by low uptake of Hoechst 33342 in vivo) contain a high proportion of long-term repopulating cells capable of serial engraftment [10,11]. These findings lead us to the hypothesis that a hypoxic microenvironment may support leukemic stem cells (LSC). It has been shown that hematopoiesis can be increased ex vivo by maintaining cell cultures at oxygen levels from 1% to 3%; in this scenario, low pO2 levels may prevent oxygen radicals from damaging HSC [12]. Little is known, however, about the role of hypoxia in the maintenance of LSC. Data from a rat leukemia model demonstrated that leukemic cells infiltrating BM reside in markedly hypoxic areas compared to cells in BM of healthy rats [13,14]. Since hypoxic tumors are in general more resistant to PLoS ONE | www.plosone.org 1 August 2011 | Volume 6 | Issue 8 | e23108