Sensitivity of acute myeloid leukemia cell lines to the dual targeting of the urokinase system and the mapk pathway by modified anthrax toxins. (c2010)

Abstract

Acute myeloid leukemia (AM L) is a hematological malignancy characterized by the rapid expansion of immature clonal myeloid cells, which leads to the failure of normal hematopoiesis. The remission rate in AML patients, following induction therapy, is 50 to 70% but most patients who undergo complete remission, recur later on with a poor prognosis. Thus leukemic re lapse in AM L remains a major therapeutic problem with the need of developing novel, tumor specific AML therapies. We aim to selectively target AM L cells using a recombinant anthrax lethal toxin (LeTx) and a urokinase-activated recombinant anthrax toxin (PrAgU2/LF). LeTx is a binary toxin consisting of 2 proteins, protective antigen (PrAg), and lethal factor (LF). PrAg binds cells through the anthrax toxin receptors (ANTXRs) and is cleaved by cell surface furin proteases leading to the release of a 20 kOa fragment and the generation of an activated 63 kOa PrAg protein. Active PrAg forms heptamers, binds 3 molecu les of LF and undergoes receptor-mediated endocytosis followed by release of LF into the cytosol. LF is a zinc metalloprotease that cleaves and inactivates mitogenactivated protein kinase (MAPK) kinases leading to the inactivation of the MAPK pathway. A modified, urokinase-activated version of anthrax lethal toxin (PrAgU2/LF) in which the furin cleavage sequence of PrAg is substituted with a sequence cleaved by the urokinase plasminogen activator (uPA/uPAR) protease system was generated. The MAPK pathway and the uPA/uPAR system have been shown to be active in a number of tumors, including AML, rendering AML cells potential targets for both LeTx (PrAg/LF) and the dual-specific, urokinase-activated PrAgU2/LF. In this study, we tested the sensitivity of a panel of AML cell lines to LeTx (PrAg/LF), PrAgU2/LF, PrAg/FP59 and PrAgU2/FP59 using a proliferation inhibition (cytotoxicity) assay. In addition, we targeted the PI3K1AKT pathway using the PI3K inhibitor LY294002 and the Ras-Raf-MEK1 /2-ERK1/2 pathway using the MEK1/2 inhibitor U0126. The majority of AML cell lines (6 out of 9) were sensitive to LeTx (PrAg/LF) with IC50 values ranging from 14 to 94 pM with a th ird of LeTx-sensitive cell lines (2 out of 6) being also sensitive to the urokinase-activated PrAgU2/LF with IC50 values of 56 and 151 pM. Furthermore, all 9 cell lines were sensitive to PrAgU2/FP59 with IC50 values ranging from 2.9 pM to 318 pM, indicating the expression of active uPA/uPAR on the surface of AML cells. In addition, treatment of LeTx-sensitive AML cells with the MEK1/2 inhibitor U0126, replicated the cytotoxicity of LeTx indicating that the cytotoxicity of LeTx (PrAg/LF) may be mediated through the inhibition of the Ras-Raf-MEK1/2-ERK1/2 branch of the MAPK pathway. AML cells that were not sensitive to LeTx were sensitive to co-incubation with the PI3K (Phosphatidylinositol 3-kinase) inhibitor L Y294002, indicating that dual inhibition of both the MAPK and PI3K1AKT pathway is necessary to target AML cells that are resistant to the inhibition of the MAPK pathway alone. These results indicate that a majority of AML cell lines are sensitive to the inhibition of the MAPK pathway and do express an active urokinase protease system rendering both these pathways attractive targets for the selective treatment of AML.