Neuropilin-1 drives tumor-specific uptake of chlorotoxin.

BACKGROUND: Chlorotoxin (Cltx) isolated from scorpion venom is an established tumor targeting and antiangiogenic peptide. Radiolabeled Cltx therapeutic (131I-TM601) yielded promising results in human glioma clinical studies, and the imaging agent tozuleristide, is under investigation in CNS cancer studies. Several binding targets have previously been proposed for Cltx but none effectively explain its pleiotropic effects; its true target remains ambiguous and is the focus of this study. METHODS: A peptide-drug conjugate (ER-472) composed of Cltx linked to cryptophycin as warhead was developed as a tool to probe the molecular target and mechanism of action of Cltx, using multiple xenograft models. RESULTS: Neuropilin-1 (NRP1), an endocytic receptor on tumor and endothelial cells, was identified as a novel Cltx target, and NRP1 binding by Cltx increased drug uptake into tumor. Metabolism of Cltx to peptide bearing free C-terminal arginine, a prerequisite for NRP1 binding, took place in the tumor microenvironment, while native scorpion Cltx with amidated C-terminal arginine did not bind NRP1, and instead acts as a cryptic peptide. Antitumor activity of ER-472 in xenografts correlated to tumor NRP1 expression. Potency was significantly reduced by treatment with NRP1 blocking antibodies or knockout in tumor cells, confirming a role for NRP1-binding in ER-472 activity. Higher cryptophycin metabolite levels were measured in NRP1-expressing tumors, evidence of NRP1-mediated enhanced drug uptake and presumably responsible for the superior antitumor efficacy. CONCLUSIONS: NRP1 was identified as a novel Cltx target which enhances tumor drug uptake. This finding should facilitate tumor selection for chlorotoxin-based therapeutics and diagnostics.

Apratoxin A Shows Novel Pancreas-Targeting Activity through the Binding of Sec 61.

Apratoxin A is a natural product with potent antiproliferative activity against many human cancer cell lines. However, we and other investigators observed that it has a narrow therapeutic window in vivo Previous mechanistic studies have suggested its involvement in the secretory pathway as well as the process of chaperone-mediated autophagy. Still the link between the biologic activities of apratoxin A and its in vivo toxicity has remained largely unknown. A better understanding of this relationship is critically important for any further development of apratoxin A as an anticancer drug. Here, we describe a detailed pathologic analysis that revealed a specific pancreas-targeting activity of apratoxin A, such that severe pancreatic atrophy was observed in apratoxin A-treated animals. Follow-up tissue distribution studies further uncovered a unique drug distribution profile for apratoxin A, showing high drug exposure in pancreas and salivary gland. It has been shown previously that apratoxin A inhibits the protein secretory pathway by preventing cotranslational translocation. However, the molecule targeted by apratoxin A in this pathway has not been well defined. By using a (3)H-labeled apratoxin A probe and specific Sec 61α/ß antibodies, we identified that the Sec 61 complex is the molecular target of apratoxin A. We conclude that apratoxin A in vivo toxicity is likely caused by pancreas atrophy due to high apratoxin A exposure. Mol Cancer Ther; 15(6); 1208-16. ©2016 AACR.

E7449: A dual inhibitor of PARP1/2 and tankyrase1/2 inhibits growth of DNA repair deficient tumors and antagonizes Wnt signaling.

Inhibition of Poly(ADP-ribose) Polymerase1 (PARP1) impairs DNA damage repair, and early generation PARP1/2 inhibitors (olaparib, niraparib, etc.) have demonstrated clinical proof of concept for cancer treatment. Here, we describe the development of the novel PARP inhibitor E7449, a potent PARP1/2 inhibitor that also inhibits PARP5a/5b, otherwise known as tankyrase1 and 2 (TNKS1 and 2), important regulators of canonical Wnt/ß-catenin signaling. E7449 inhibits PARP enzymatic activity and additionally traps PARP1 onto damaged DNA; a mechanism previously shown to augment cytotoxicity. Cells deficient in DNA repair pathways beyond homologous recombination were sensitive to E7449 treatment. Chemotherapy was potentiated by E7449 and single agent had significant antitumor activity in BRCA-deficient xenografts. Additionally, E7449 inhibited Wnt/ß-catenin signaling in colon cancer cell lines, likely through TNKS inhibition. Consistent with this possibility, E7449 stabilized axin and TNKS proteins resulting in ß-catenin de-stabilization and significantly altered expression of Wnt target genes. Notably, hair growth mediated by Wnt signaling was inhibited by E7449. A pharmacodynamic effect of E7449 on Wnt target genes was observed in tumors, although E7449 lacked single agent antitumor activity in vivo, a finding typical for selective TNKS inhibitors. E7449 antitumor activity was increased through combination with MEK inhibition. Particularly noteworthy was the lack of toxicity, most significantly the lack of intestinal toxicity reported for other TNKS inhibitors. E7449 represents a novel dual PARP1/2 and TNKS1/2 inhibitor which has the advantage of targeting Wnt/ß-catenin signaling addicted tumors. E7449 is currently in early clinical development.

Porcine endogenous retrovirus transmission characteristics of galactose alpha1-3 galactose-deficient pig cells.

Galactose alpha1-3 galactose (Gal) trisaccharides are present on the surface of wild-type pig cells, as well as on viruses particles produced from such cells. The recognition of Gal sugars by natural anti-Gal antibodies (NAb) in human and Old World primate serum can cause the lysis of the particles via complement-dependent mechanisms and has therefore been proposed as an important antiviral mechanism. Recently, pigs have been generated that possess disrupted galactosyl-transferase (GGTA1) genes. The cells of these pigs do not express Gal sugars on their surface, i.e., are Gal null. Concerns have been raised that the risk of virus transmission from such pigs may be increased due to the absence of the Gal sugars. We investigated the sensitivity of porcine endogenous retrovirus (PERV) produced from Gal-null and Gal-positive pig cells to inactivation by purified NAb and human serum. PERV produced in Gal-null pig cells was resistant to inactivation by either NAb or human serum. In contrast, although Gal-positive PERV particles were sensitive to inactivation by NAb and human serum, they required markedly higher concentrations of NAb for inactivation compared to the Gal-positive cells from which they were produced. Complete inactivation of Gal-positive PERV particles was not achievable despite the use of high levels of NAb, indicating that NAb-mediated inactivation of cell-free PERV particles is an inefficient process.

Production of alpha-1,3-galactosyltransferase null pigs by means of nuclear transfer with fibroblasts bearing loss of heterozygosity mutations.

Hyperacute rejection of porcine organs by old world primate recipients is mediated through preformed antibodies against galactosyl-alpha-1,3-galactose (Galalpha-1,3-Gal) epitopes expressed on the pig cell surface. Previously, we generated inbred miniature swine with a null allele of the alpha-1,3-galactosyltransferase locus (GGTA1) by nuclear transfer (NT) with gene-targeted fibroblasts. To expedite the generation of GGTA1 null pigs, we selected spontaneous null mutant cells from fibroblast cultures of heterozygous animals for use in another round of NT. An unexpectedly high rate of spontaneous loss of GGTA1 function was observed, with the vast majority of null cells resulting from loss of the WT allele. Healthy piglets, hemizygous and homozygous for the gene-targeted allele, were produced by NT by using fibroblasts that had undergone deletional and crossover/gene conversion events, respectively. Aside from loss of Galalpha-1,3-Gal epitopes, there were no obvious phenotypic differences between these null piglets and WT piglets from the same inbred lines. In fact, congenital abnormalities observed in the heterozygous NT animals did not reappear in the serially produced null animals.

Expression of xenogeneic MHC class II molecules in HLA-DR(+) and -DR(-) cells: influence of retrovirus vector design and cellular context.

We recently established that molecular chimeras of major histocompatibility complex (MHC) class II molecules, created via retroviral transfer of allogeneic class II cDNAs into bone marrow cells (BMCs), alleviated complications associated with mixed BMC chimeras while leading to T cell tolerance to renal grafts sharing the transferred class II. Initially demonstrated for allogeneic transplants in miniature swine, this concept was extended to T-dependent antibody (Ab) responses to xenogeneic antigens (Ags) in the pig --> baboon combination. Successful down-regulation of T cell responses appeared, however, to be contingent on a tight lineage-specific expression of transferred class II molecules. The present studies were, therefore, designed to evaluate the influence of construct design and cellular environment on expression of retrovirally transferred xenogeneic class II cDNAs. Proviral genomes for pig class II SLA-DR expression, differing only at the marker neo(r) or enhanced green fluorescent protein (EGFP) gene, showed increased membrane SLA-DR density on HLA-DR(-) fibroblasts as well as HLA-DR(+), TF-1 erythroleukemia cells. More importantly, HLA-DR(+) human B cell lines, although efficiently transduced with pig DR retroviruses, exhibited unstable surface pig DR. Surface pig DR- B cells, nevertheless, stimulated autologous human T cells pre-sensitized to pig Ags, a proliferation likely occurring through presentation of class II-derived peptides. Collectively, these data suggest that surface expression of transferred class II molecules is not related to the ability of recipient cells to synthesize xenogeneic class II molecules but rather to their Ag processing capacities.

Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning.

The presence of galactose alpha-1,3-galactose residues on the surface of pig cells is a major obstacle to successful xenotransplantation. Here, we report the production of four live pigs in which one allele of the alpha-1,3-galactosyltransferase locus has been knocked out. These pigs were produced by nuclear transfer technology; clonal fetal fibroblast cell lines were used as nuclear donors for embryos reconstructed with enucleated pig oocytes.