Abstract 4641: Hypoxia activated pro-drug TH-302 induces hypoxia-dependent anti-leukemia activity in vitro and in vivo
Authors: Benito Juliana M. , Lu Hongbo , Shi Yuexi , Jacamo Rodrigo O. , McQueen Teresa J. , Konoplev Sergej , Bueso-Ramos Carlos , Zweidler-McKay Patrick , Handisides Damian , Hart Charles P. , Andreeff Michael , Konopleva Marina 
Published: August 2012

Abstract
Hypoxia in tumors is generally associated with chemoresistance and radioresistance. Hypoxia-inducible factor-1α (HIF-1α) was expressed in the bone marrow in 58% of primary acute myelogenous (AML) cases by immunohistochemistry (n=156). We have recently demonstrated a marked expansion of hypoxia in leukemia bone marrow (Benito PLoSOne 2011) and demonstrated hypoxia promotes chemoresistance in acute leukemias. Normoxic stabilization of HIF-1α using inhibitor of prolyl hydroxylases DMOG, or overexpression through infection with lentiviral non-degradable mutant HIF-1α, significantly diminished apoptosis induced by vincristine and etoposide in REH and Nalm-6 cells. These findings suggest that hypoxia mediates chemoresistance at least in part through HIF-1a. TH-302 is a 2-nitroimidazole of the cytotoxin bromo-isophosphoramide mustard that upon hypoxia-dependent activation induces DNA alkylation, a novel approach to target leukemia. We report the in vitro and in vivo anti leukemia activity of TH-302. In vitro, TH-302 exhibited potent anti-leukemia activity in pre-B ALL (REH, NALM6), AML (OCI-AML3, MOLM-13, KG-1) and CML cell lines (KBM5), with IC50s at 1% O2 ranging from 0.04µM to 2.3µM. The hypoxic cytotoxicity ratios (aerobic IC50/hypoxic IC50) were 11 and 92 in ALL (REH, NALM6) and 14, 80 and 116 in AML (OCI-AML3, KG-1, KBM5). The drug at 5-7.5µM also exhibited specific hypoxia dependent anti-leukemia activity in primary ALL and AML samples (normoxic vs hypoxic specific apoptosis defined as %AnnV positive cells +/− SEM normalized to untreated control cells): ALL-1: 5.1+/−0.5% vs 65.3+/−4.2%; ALL-2: 8.3+/−1.9% vs 28.4+/−6.3%; AML 1: 2.6+/−0.2% vs 40.3+/−1.5%). In in vivo leukemia models TH-302 (50 mg/kg IP 3 times a week for three weeks) reduced the number of circulating AML cells (control, 13.2+/−5.7 x106/ml; TH-302, 2.5+/−2.1 x106/ml) and prolonged survival of NSG mice engrafted with primary AML cells compared to the vehicle treated mice (median survival time: TH-302=75 days; Control=56 days; P=0.003). Finally, anti-leukemia efficacy of TH-302 was tested in vitro in combination with chemotherapy commonly used for treatment of AML (AraC, doxorubicin) and with demethylating agents azacitidine (5-AZA) and decitabine (DAC). Combination TH-302 with AraC or with DAC were synergistic in killing OCI-AML3 and MOLM-13 AML cells under hypoxia (combination index values of 0.4 and 0.67 for AraC plus TH-302 in MOLM13 and OCI-AML3, respectively; 0.3 for DAC plus TH-302 in OCI-AML3). In summary, our findings suggest that hypoxia and HIF-1α constitute important factors in the survival of leukemic blasts within the BM microenvironment. The results support targeting hypoxia with a hypoxia-activated drug such as TH-302 to enhance the efficacy of therapeutic regimens in AML. A Phase 1 clinical study of TH-302 in patients with relapsed/refractory hematologic malignancies is ongoing.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4641. doi:1538-7445.AM2012-4641