Sensitivity of acute myeloid leukemia cells to the dual targeting of the urokinase plasminogen activator and the MAPK pathway by a urokinase-activated anthrax lethal toxin. (c2013)

Abstract

Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by abnormal proliferation of myeloid progenitor cells. Around 15,000 new case of AML are diagnosed each year with a fatality rate of 65%. Conventional treatment consists of two phases, an induction phase and a consolidation phase. Though standard treatment yields a high remission rate, the majority of patients eventually relapse due to the proliferation of drug-resistant leukemic blasts in the bone marrow, hence the need for alternative approaches employing novel, more selective mechanisms for targeting AML blasts. In this study, we attempt to target both the MAPK pathway and the uPA/uPAR protease system in AML cells using a dual-selective, urokinase activated recombinant anthrax lethal toxin (PrAgU2/LF). Anthrax lethal toxin (PrAg/LF) is a binary toxin that consists of two separate proteins, a protective antigen (PrAg) and a lethal factor (LF). PrAg binds cells, is cleaved by furin, oligomerizes, binds three to four molecules of LF, and undergoes endocytosis, releasing LF into the cytosol. LF cleaves MAPK kinases, inhibiting the MAPK pathway. We have generated a modified anthrax lethal toxin in which the furin activation site was replaced with uPA/uPAR activation site generating a urokinase-targeted anthrax lethal toxin, PrAgU2/LF. The MAKP pathway is a conserved pathway that regulates growth, proliferation, differentiation, and death. Its constitutive activation promotes proliferation and survival of most human cancer cells. The urokinase plasminogen activator is a serine protease consisting of the urokinase plasminogen activator (uPA) and its receptor (uPAR) and it has been shown to be overexpressed in a wide array of solid and hematological malignancies including AML. We tested the potency and selectivity of PrAgU2/LF on a panel of 11 human AML cell lines and two normal hematopoietic cells, peripheral mononuclear cells and CD34 + progenitor bone marrow blasts. Five AML cell lines showed cytotoxic responses to PrAgU2/LF while none of the normal cells tested were targeted by this toxin indicating potency and selectivity of this dual-selective molecule in AML. Further analysis revealed that, out of the 6 AML cell lines that did not respond to PrAgU2/LF, 4 were also not responsive to the wild-type PrAg/LF, indicating resistance to the inhibition of the MAPK pathway, while the remaining 2 cell lines did not express the uPA/uPAR system. Furthermore, sensitivity of cells to PrAgU2/LF was linked to the expression levels of uPAR and was reversed through the inhibition of the uPA/uPAR system. Finally, a dose escalation study in mice showed that the maximal tolerated dose (MTD) of PrAgU2/LF is more than 4-fold higher than that of the wild-type PrAg/LF. Hence, introduction of the urokinase-activation sequence generated a dual-selective molecule whose activity requires both the expression of the uPA/uPAR system and the dependence to the MAPK pathway by the targeted cells. In this study, we have shown that the Urokinase-activated anthrax lethal toxin (PrAgU2/LF) selectively targets AML cells while sparing normal blasts and is a novel, dual selective molecule for the potential targeting of AML.