The limited efficacy of the current antitumor microenvironment strategies is due in part to the poor understanding of the roles and relative contributions of the various tumor stromal cells to tumor development. Here, we describe a versatile in vivo anthrax toxin protein delivery system allowing for the unambiguous genetic evaluation of individual tumor stromal elements in cancer. Our reengineered tumor-selective anthrax toxin exhibits potent antiproliferative activity by disrupting ERK signaling in sensitive cells. Since this activity requires the surface expression of the capillary morphogenesis protein-2 (CMG2) toxin receptor, genetic manipulation of CMG2 expression using our cell-type–specific CMG2 transgenic mice allows us to specifically define the role of individual tumor stromal cell types in tumor development. Here, we established mice with CMG2 only expressed in tumor endothelial cells (ECs) and determined the specific contribution of tumor stromal ECs to the toxin’s antitumor activity. Our results demonstrate that disruption of ERK signaling only within tumor ECs is sufficient to halt tumor growth. We discovered that c-Myc is a downstream effector of ERK signaling and that the MEK–ERK–c-Myc central metabolic axis in tumor ECs is essential for tumor progression. As such, disruption of ERK–c-Myc signaling in host-derived tumor ECs by our tumor-selective anthrax toxins explains their high efficacy in solid tumor therapy.

FDA-approved BRAF and MEK small molecule inhibitors have demonstrated some level of efficacy in patients with metastatic melanomas. However, these “targeted” therapeutics have a very low therapeutic index, since these agents affect normal cells, causing undesirable, even fatal, side effects. To address these significant drawbacks, here, we have reengineered the anthrax toxin-based protein delivery system to develop a potent, tumor-selective MEK inactivator. This toxin-based MEK inactivator exhibits potent activity against a wide range of solid tumors, with the highest activity seen when directed towards tumors containing the BRAFV600E mutation. We demonstrate that this reengineered MEK inactivator also exhibits an extremely high therapeutic index (>15), due to its in vitro and in vivo activity being strictly dependent on the expression of multiple tumor-associated factors including tumor-associated proteases matrix metalloproteinase, urokinase plasminogen activator, and anthrax toxin receptor capillary morphogenesis protein-2. Furthermore, we have improved the specificity of this MEK inactivator, restricting its enzymatic activity to only target the ERK pathway, thereby greatly diminishing off-target toxicity. Together, these data suggest that engineered bacterial toxins can be modified to have significant in vitro and in vivo therapeutic effects with high therapeutic index. Significance Many naturally occurring bacterial protein toxins have evolved to target cellular signaling pathways that are often dysregulated to drive progression of human cancers. As such, bacterial toxins serve as a potential valuable source for novel anti-tumor agents. Here, through reengineering the unique structural/functional domains of anthrax toxin, we describe the successful development of a potent, highly tumor-selective MEK inactivator, which is effective against a wide variety of tumors with an extremely high therapeutic index, thereby demonstrating clear advantages in both potency and specificity over the current FDA-approved small-molecule inhibitors. Importantly, we demonstrate that the immunogenicity of the toxin can be safely and efficiently overcome by a B-cell depleting regimen. These observations suggest that bacterial toxins can be re-engineered/re-purposed to target human tumors.