Mechanisms of Cytotoxicity of Wild-Type Anthrax Lethal Toxin (PrAg/LF) to Acute Myeloid Leukemia Cells

Abstract

Anthrax lethal toxin (PrAg/LF) is a binary toxin composed of protective antigen PrAg and lethal toxin LF utilized as a potential targeted therapeutic against cancers including acute myeloid leukemia (AML). To understand PrAg/LF anti-tumor activity, response mechanisms need to be evaluated. One response to extracellular stressors is autophagy. Autophagy is an evolutionary conserved mechanism inherent in normal cells that allows adaptation to physical, chemical and biological stressors. Intracellular contents are recycled and degraded where they are delivered to the lysosome. This process is strictly regulated, and dysregulation is seen in pathological conditions such as cancer. In cancer, autophagy is considered as a double-edged sword exhibiting both tumor protective and tumor suppressive roles. In this study, we aimed to examine the effects of treating a panel of 7 acute myeloid leukemia (AML) cell lines with PrAg/LF in the context of autophagy. We saw that autophagosome formation using Cyto-ID is seen in all 7 cell lines upon PrAg/LF treatment regardless of their sensitivity to our drug. Then we detected upstream regulators of autophagy using Flow Cytometry intracellular staining for p-ULK. This showed a complex temporal initiation of autophagy activation suggesting autophagy can be time and cell specific. Then cytotoxicity assays were done to assess autophagy’s role in cell death by using chloroquine (CQ) as an autophagy inhibitor. In 3 sensitive cell lines, we report autophagy as a deleterious process in PrAg/LF induced cell death. On the other hand, one sensitive cell line exhibited a delay in cell death when autophagy was not inhibited. The resistant cell lines that displayed no cell death and autophagy activation in the Cyto-ID and p-ULK detection levels results may suggest that PrAg/LF can initiate autophagy as a cellular response, but it is not a determinant factor in predicting cell survival. This proposes that the two resistant cell lines might rely on other survival mechanisms for their maintenance. The importance of characterizing the effects of autophagy under targeted therapy is crucial in determining possible therapeutic implications.