Characterization of the potency and mechanism of action of a novel dual-selective intermolecularly complemented version of Anthrax Lethal Toxin (PAU2R200A- PAL1I207R/FP59) in Acute myeloid leukemia

Abstract

Anthrax lethal toxin (PrAg/LF) is a binary toxin consisting of protective antigen (PrAg), the cell binding moiety, and lethal factor (LF), the catalytic moiety. Inside the cell, LF cleaves MEKs and subsequently leads to the inhibition of the MAPK pathway causing cell death. Due to its off-target toxicity, a more selective generation of PrAg/LF was re-engineered by making its activation dependent on cleavage by tumor specific cell surface proteases enriched on the surface of tumor cells and not normal cells. This resulted in PrAgU2 and PrAgL1 variants that mandate activation by either uPA/uPAR or MMPs tumor specific proteases, respectively. Both variants proved to be highly potent against tumors while having an enhanced selectivity and as such paved the way to create a modified intermolecularly complementing (IMC), PAU2R200A-PAL1I207R, version of PrAg that requires activation by both uPA/uPAR and MMPs proteases, simultaneously. IMC combined with LF or FP59, an inhibitor of protein synthesis, is thought to be highly selective requiring two distinct proteolytic activities overexpressed by tumor tissues for its activation. In this study we tested the potency of IMC/FP59 and IMC/LF on a panel of AML cell lines. IMC/LF treatment didn’t show any signs of cytotoxicity to AML cells, but induced cell cycle arrest in a subset of these cells. On the other hand, IMC/FP59 displayed potency on AML cells with four levels of sensitivity seen; high sensitivity, moderate sensitivity, mild sensitivity, and no sensitivity. We showed evidence that PAU2R200A/FP59, from the IMC variant, induced a cytotoxic response that matched the pattern of IMC/FP59, as such indicating that uPA/uPAR is the rate limiting factor in the activation of IMC/FP59.In addition, staining for Annexin V/PI post IMC/FP59 treatment showed an increase in double positive cells indicating non-apoptotic cell death. The sensitivity of AML cells to IMC/FP59 did not depend on the basal levels of expression of uPAR and MMPs (MMP2 and MMP9). However, given that MMP9 was found not to be expressed in the sensitive cell lines, we were able to exclude its expression as a requirement for cytotoxicity. The absence of dependence indicated that cytotoxicity levels seen may depend on the activity levels of these proteases, rather than on their expression levels. Finally, while IMC/LF treatment did not affect the expression of any of the proteases tested (uPAR, MMP2, and MMP9), treatment with IMC/FP59 did affect their expression with significant cytotoxic responses seen only in cells whose uPAR expression was not affected at any time point post-treatment. In this study, we showed for the first time the potency of IMC PrAg variant on AML cell lines, its mode of action, as well as its mechanism of cell death.