Cnf1 Variants Endowed with the Ability to Cross the Blood–Brain Barrier: A New Potential Therapeutic Strategy for Glioblastoma

Abstract

Among gliomas, primary tumors originating from glial cells, glioblastoma (GBM) identified as WHO grade IV glioma, is the most common and aggressive malignant brain tumor. We have previously shown that the Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1) is remarkably effective as an anti-neoplastic agent in a mouse model of glioma, reducing the tumor volume, increasing survival, and maintaining the functional properties of peritumoral neurons. However, being unable to cross the blood–brain barrier (BBB), CNF1 requires injection directly into the brain, which is a very invasive administration route. Thus, to overcome this pitfall, we designed a CNF1 variant characterized by the presence of an N-terminal BBB-crossing tag. The variant was produced and we verified whether its activity was comparable to that of wild-type CNF1 in GBM cells. We investigated the signaling pathways engaged in the cell response to CNF1 variants to provide preliminary data to the subsequent studies in experimental animals. CNF1 may represent a novel avenue for GBM therapy, particularly because, besides blocking tumor growth, it also preserves the healthy surrounding tissue, maintaining its architecture and functionality. This renders CNF1 the most interesting candidate for the treatment of brain tumors, among other potentially effective bacterial toxins.

Mobilizing Toxins for Cancer Treatment: Historical Perspectives and Current Strategies

“…Intravenous administration of the An2-CNF1-H8 variant upregulates spinophilin in the mouse hippocampus, suggesting BBB bypass. Altogether, these results demonstrate that the An2-CNF1-H8 variant is likely able to cross the BBB to induce cell death in GBM cells [4] and may be translated to future clinical studies.…”

“…The anticoagulants tyrofabin and hirudin, for example, originate from venom of the African saw-scaled viper and leech secretions, respectively [1]. Even pathogenic bacteria typically considered harmful to healthy tissue may prove to be clinically useful as studies have shown that toxins produced by these organisms can be manipulated to target aberrant cells in a tissue-or cell-specific manner [2][3][4][5].…”

“…Escherichia coli protein toxin, cytotoxic necrotizing factor 1 (CNF1), acts as an effective anti-neoplastic in glioma mouse models, reducing tumor volume and increasing survival, all while preserving the functional properties of the surrounding neurons [22,23]. As one paper acknowledges, therapies against glioma cells must be able to cross the blood-brain barrier (BBB), otherwise, treatment would have to be directly injected into the brain [4]. To circumvent invasive cranial injections, CNF1 was reengineered with an N-terminal BBB-crossing tag.…”

CTX-CNF1 Recombinant Protein Selectively Targets Glioma Cells In Vivo

“…As a matter of fact, toxins are extremely effective enzymes with great selectivity towards specific cellular substrates and, importantly, they are not subjected to drug resistance mechanisms [ 20 ]. Furthermore, they can be easily modified: their active core can be isolated and cloned to more efficiently penetrate into solid tumors, or they can be combined with carriers and antibodies to specifically enter cancer cells [ 4 , 5 ].…”

“…When administered in vivo, CNF1 increases the survival of glioma-bearing mice and enhances neuronal function and plasticity, sparing neuronal responses in peritumoral areas [ 2 , 3 , 4 ]. Despite the promising results obtained in preclinical models, CNF1 translatability to clinics is limited by its inability of crossing the blood–brain barrier (BBB) in normal conditions [ 5 ]. Recent studies have proven that Chlorotoxin (CTX), a 36 amino acid peptide derived from the venom of the scorpion Leiurus quinquestriatus, is able to penetrate the BBB and to selectively recognize and target glioma cells [ 6 , 7 , 8 , 9 ].…”