Anthrax toxin is a three-part toxin secreted by Bacillus anthracis, consisting of three components: protective antigen (PA), lethal factor (LF), and edema factor (EF). Among the three components of the toxin, PA is the cellular binding moiety, which binds to its cellular receptors Tumor Endothelium Marker-8 (TEM8) and Capillary Morphogenesis Protein-2 (CMG2). Upon binding to target cell surface, PA is proteolytically activated by the ubiquitously expressed cell surface furin protease, resulting in the formation of active PA oligomer, which in turn binds and translocates the two enzymatic moieties LF and EF into the cytosol of cells. LF, which forms lethal toxin (LT) with PA, cleaves several mitogen-activated protein kinase kinases (MEKs) and rodent inflammasome sensor Nlrp1. EF, which forms edema toxin (ET) with PA, is an adenylate cyclase that generates abnormally high concentrations of cAMP. LT and ET are two of the major virulence factors of B. anthracis. The absolute requirement for proteolytic activation of PA provided us the opportunity to reengineer PA to be selectively activated by tumor-associated proteases, generating tumor-targeting toxins. We have previously designed and characterized variants of anthrax lethal toxin that are selectively activated by either matrix-metalloproteinases (MMPs) or urokinase plasminogen activator (uPA) activities. These tumor-selective anthrax toxins are cytotoxic in the tumor microenvironment and display broad and potent anti-tumor activities in vivo. In the year of 2014, by collaborating with the Bugge lab in NIDCR, we have performed the first direct comparison of the safety and efficacy of three engineered anthrax lethal toxin variants requiring activation by MMPs, uPA, or co-localized MMP/uPA activities. LF, which can specifically disrupt the RAS-RAF-MEK-ERK pathway, was used as the effector toxin in this study. C57BL/6J mice were challenged with six doses of engineered toxins via intraperitoneal (I.P.) or intravenous (I.V.) dose routes to determine the maximum tolerated dose for six administrations (MTD6) and dose-limiting toxicities. Efficacy was evaluated using the B16-BL6 syngraft model of melanoma;mice bearing established tumors were treated with six I.P. doses of toxin and tumor measurements and immunohistochemistry, paired with terminal blood work, were used to elaborate upon the anti-tumor mechanism and relative efficacy of each variant. We found that MMP-, uPA- and dual MMP/uPA-activated anthrax lethal toxins exhibited the same dose-limiting toxicity;dose-dependent GI toxicity. In terms of efficacy, all three toxins significantly reduced primary B16-BL6 tumor burden, ranging from 32% to 87% reduction, and they also delayed disease progression as evidenced by dose-dependent normalization of blood work values. While target organ toxicity and effective doses were similar amongst the variants, the dual MMP/uPA-activated anthrax lethal toxin exhibited the highest I.P. MTD6 and was 1.5-3-fold better tolerated than the single MMP- and uPA-activated toxins. Overall, we demonstrate that this dual MMP/uPA-activated anthrax lethal toxin can be administered safely and is highly effective in a preclinical model of melanoma. This modified bacterial cytotoxin is thus a promising candidate for further clinical development and evaluation for use in treating human cancers. In addition to LF, other effector proteins, such as cytolethal distending toxin (Cdt), can also be delivered to tumor cells using the tumor-selective PA variants described above. Cdt is produced by Gram-negative bacteria of several species, including the human pathogens Escherichia coli, Haemophilus ducreyi, Aggregatibacter actinomycetemcomitans, Campylobacter jejuni, and Shigella dysenteriae. Cdt is composed of three subunits, CdtA, CdtB, and CdtC, with CdtB being the catalytic subunit. In the year of 2014, we fused CdtB from H. ducreyi to the N-terminal 255 amino acids of LF to design a novel, potentially potent antitumor drug. As a result of this fusion, CdtB was transported into the cytosol of targeted cells via the efficient delivery mechanism of anthrax toxin. The fusion protein efficiently killed various human tumor cell lines by first inducing a complete cell cycle arrest in the G2/M phase, followed by induction of apoptosis. The fusion protein showed very low toxicity in mouse experiments and impressive antitumor effects in a Lewis Lung carcinoma model, with a 90% cure rate. This study demonstrates that efficient drug delivery by a modified anthrax toxin system combined with the enzymatic activity of CdtB has great potential as anticancer treatment and should be considered for the development of novel anticancer drugs. Recently, we found that LF can also cleave Nlrp1 proteins from certain strains of rats and mice at sites near their amino-termini, resulting in activation of the NLRP1 inflammasomes, eliciting a caspase 1-mediated rapid macrophage cell death, termed pyroptosis. Nlrp1 encodes the NOD-like receptor (NLR) protein NLRP1, the sensor component of the NLRP1 inflammasome, a multiprotein complex responsible for activation of caspase-1. In the year of 2014, we performed a screen of NF-κB inhibitors for protection against anthrax LT-induced macrophage pyroptosis. We identified NaAsO2 as a potent inhibitor of NLRP1 inflammasome. It protected both primary macrophages (from BALB/cJ mice) and macrophage cell line RAW264.7 from anthrax LT over a range of concentrations. We discovered that other arsenicals, including As2O3, a U.S. Food and Drug Administration approved drug for the treatment of APL, multiple myeloma, and other myelodysplastic syndromes, also protected against LT intoxication. Importantly, these arsenicals not only inhibited activation of the NLRP1 inflammasome but also NLRP3 and NAIP5/NLRC4 inflammasomes by their respective activating signals, anthrax lethal toxin, nigericin, and flagellin. These compounds prevented the autoproteolytic activation of caspase-1 and the processing and secretion of IL-1-Beta from macrophages. Inhibition was independent of protein synthesis induction, proteasome-mediated protein breakdown, or kinase signaling pathways. Arsenic trioxide and sodium arsenite did not directly modify or inhibit the activity of preactivated recombinant caspase-1. Rather, they induced a cellular state inhibitory to both the autoproteolytic and substrate cleavage activities of caspase-1, which was reversed by the reactive oxygen species scavenger N-acetylcysteine but not by reducing agents or NO pathway inhibitors. Arsenicals provided protection against NLRP1-dependent anthrax lethal toxin-mediated cell death and prevented NLRP3-dependent neutrophil recruitment in a monosodium urate crystal inflammatory murine peritonitis model. These findings suggest a novel role in inhibition of the innate immune response for arsenical compounds that have been used as therapeutics for several hundred years.
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