Characterization of the Cytotoxic Mechanism and Mode of Cell Death Induced by a MEK1/2-selective version of Anthrax Lethal Toxin (PrAg/LFW271A) in Acute Myeloid Leukemia

Abstract

Wild-type anthrax lethal toxin (PrAg/LF) demonstrated high potency in inhibiting the MAPK pathway in AML cell lines that rely on this pathway for survival. PrAg/LF is a binary toxin consisting of protective antigen (PrAg), that serves as the tumor specific delivery protein, and the wild-type effector LF, which inhibits three crucial MAPK pathways —ERK, P38, and JNK—by cleaving MEK1/2, MEK3/6, and MEK4/7, respectively. Although PrAg/LF exhibits tumor selectivity, its in vivo toxicity restricts its clinical application. Based on the finding that the potency of LF in inhibiting tumor growth is primarily attributed to its MEK1/2 inactivation properties, a novel MEK1/2-selective version of Anthrax Lethal Toxin (PrAg/LFW271A) that spares the non-specific inhibition of other MKKs such as the p38 and JNK pathways, was developed. In this study, we aimed to examine the anti-tumor activity, the cytotoxic mechanism, and the mode of cell death of the new variant PrAg/LFW271A. The results from the cytotoxicity assays exhibit similar anti-tumor activity between PrAg/LF and PrAg/LFW271A in 4 out of 7 human AML cell lines. The ERK pathway examination revealed inhibition of this pathway in sensitive and resistant cells. This indicates the reliance of the sensitive cells on the ERK pathway, while the resistance mechanism might rely on other pathways for survival. Additionally, staining for Annexin V/propidium iodide following treatment showed no evidence for apoptotic death following ERK pathway inhibition. Furthermore, we showed activation of autophagy in a number of cell lines following treatment with both the wild-type and MEK1/2-specific version of LF. Inhibition of autophagy using CQ, abrogated both the LF- and the LFW271A-mediated cell death in a number of AML cell line, indicating that, in these cells, the inhibition of the MAPK pathway leads to autophagy-mediated cell death (death by autophagy). In this study, we have shown that PrAg/LFW271A maintained the same anti-tumor activity as the wild-type PrAg/LF by inhibiting the ERK pathway, which led to autophagy activation and autophagic cell death in a subset of AML cells.