Dual Targeting of Matrix Metalloproteases and the MAPK Pathway in Acute Myeloid Leukemia Cells Using an MMP-activated anthrax lethal toxin (PrAgL1/LF)

Abstract

Anthrax lethal toxin is a two-component toxin consisting of protective antigen (PrAg), the cell binding and translocation domain, and lethal factor (LF), the catalytic domain, which cleaves all mitogen-activated protein/extracellular regulated kinase kinases (MEKs), leading to the inhibition of the MAPK pathway. In order to increase selectivity of this toxin, we have replaced the furin cleavage site with a matrix metalloprotease cleavage site generating an MMP-activated anthrax lethal toxin; PrAgL1/LF. In this study, we attempt to target both the mitogen-activated protein kinase pathway and matrix metalloproteases in acute myeloid leukemia (AML) cell lines. Additionally, we also determine the level of autophagy activation, through quantification of autophagosomes on flow cytometry, and the impact of its inhibition, using the autophagy inhibitor chloroquine (CQ), on cell death secondary to MAPK inhibition. Moreover, we attempt to vertically inhibit the MAPK pathway in AML cell lines in order to overcome acquired resistance of AML cell lines to PrAg/LF by targeting the MAPKK protein MEK-1/2 as well as the MAPKKK protein ERK-1/2 via SCH772984, showing that coupling the two inhibitors may offer a novel targeting method in order to overcome acquired resistance to MEK-1/2 inhibitors. Our results demonstrate that vertical inhibition does offer increased potency in comparison to single targeting of the MAPK pathway when working with sensitive cell lines that are dependent on the cascade. Several cell lines tested were sensitive to PrAgL1/LF indicating that they express MMPs and are sensitive to the inhibition of the MAPK pathway. The expression of MMPs by AML cell lines was further confirmed by their sensitivity to PrAgL1/FP59, an MMP activated version of anthrax toxin whose cytotoxicity is not related to their dependence on the MAPK pathway. Inhibition of the MAPK pathway through PrAgL1/LF led to sustained activation of autophagy, starting at 24 hours and lasting up to 120 hours following treatment with PrAgL1/LF in the AML cell lines. Addition of CQ led to an increase in cell cytotoxicity following treatment with PrAgL1/LF, at all time points, indicating that the inhibition of the MAPK pathway in AML cells leads to the activation of autophagy, which in turn mediates cell death following treatment with PrAgL1/LF. Our study shows that AML cells express MMPs and can be targeted using an MMP-activated anthrax lethal toxin and that the LF-mediated inhibition of the MAPK pathway activates autophagy which is protective at later time points.