Steve H. Thorne

Chemical control of protein stability and function in living mice

Conditional control of protein function in vivo offers great potential for deconvoluting the roles of individual proteins in complicated systems. We recently developed a method in which a small protein domain, termed a destabilizing domain, confers instability to fusion protein partners in cultured cells. Instability is reversed when a cell-permeable small molecule binds this domain. Here we describe the use of this system to regulate protein function in living mammals. We show regulation of secreted proteins and their biological activity with conditional secretion of an immunomodulatory cytokine, resulting in tumor burden reduction in mouse models. Additionally, we use this approach to control the function of a specific protein after systemic delivery of the gene that encodes it to a tumor, suggesting uses for enhancing the specificity and efficacy of targeted gene-based therapies. This method represents a new strategy to regulate protein function in living organisms with a high level of control.

Directed Evolution Generates a Novel Oncolytic Virus for the Treatment of Colon Cancer

Background Viral-mediated oncolysis is a novel cancer therapeutic approach with the potential to be more effective and less toxic than current therapies due to the agents selective growth and amplification in tumor cells. To date, these agents have been highly safe in patients but have generally fallen short of their expected therapeutic value as monotherapies. Consequently, new approaches to generating highly potent oncolytic viruses are needed. To address this need, we developed a new method that we term “Directed Evolution” for creating highly potent oncolytic viruses. Methodology/Principal Findings Taking the “Directed Evolution” approach, viral diversity was increased by pooling an array of serotypes, then passaging the pools under conditions that invite recombination between serotypes. These highly diverse viral pools were then placed under stringent directed selection to generate and identify highly potent agents. ColoAd1, a complex Ad3/Ad11p chimeric virus, was the initial oncolytic virus derived by this novel methodology. ColoAd1, the first described non-Ad5-based oncolytic Ad, is 2–3 logs more potent and selective than the parent serotypes or the most clinically advanced oncolytic Ad, ONYX-015, in vitro. ColoAd1s efficacy was further tested in vivo in a colon cancer liver metastasis xenograft model following intravenous injection and its ex vivo selectivity was demonstrated on surgically-derived human colorectal tumor tissues. Lastly, we demonstrated the ability to arm ColoAd1 with an exogenous gene establishing the potential to impact the treatment of cancer on multiple levels from a single agent. Conclusions/Significance Using the “Directed Evolution” methodology, we have generated ColoAd1, a novel chimeric oncolytic virus. In vitro, this virus demonstrated a >2 log increase in both potency and selectivity when compared to ONYX-015 on colon cancer cells. These results were further supported by in vivo and ex vivo studies. Furthermore, these results have validated this methodology as a new general approach for deriving clinically-relevant, highly potent anti-cancer virotherapies.

Using in Vivo Bioluminescence Imaging to Shed Light on Cancer Biology

Luciferases are light-emitting enzymes that have been used as reporters of biological function for several decades, and have more recently been used as reporters for the study of biological processes in living animals. Although these enzymes appear to have evolved independently in different species, they are all oxygenases that require energy, a chemical substrate, and oxygen. The technologies of detecting their weak bioluminescent signals in the bodies of living rodent models of human biology and disease, comprise the optical imaging method called in vivo bioluminescence imaging (BLI). BLI has been applied to a number of questions in cancer research, including studies of tumor burden, response to therapy, assessment of gene expression, and development of metastatic lesions. The considerations necessary for evaluating image data obtained by this method, the advances in technology development, and recent applications in the study of cancer are the focus of this paper.

CNOB/ChrR6, a new prodrug enzyme cancer chemotherapy

We report the discovery of a new prodrug, 6-chloro-9-nitro-5-oxo-5H-benzo(a)phenoxazine (CNOB). This prodrug is efficiently activated by ChrR6, the highly active prodrug activating bacterial enzyme we have previously developed. The CNOB/ChrR6 therapy was effective in killing several cancer cell lines in vitro. It also efficiently treated tumors in mice with up to 40% complete remission. 9-Amino-6-chloro-5H-benzo(a)phenoxazine-5-one (MCHB) was the only product of CNOB reduction by ChrR6. MCHB binds DNA; at nonlethal concentration, it causes cell accumulation in the S phase, and at lethal dose, it induces cell surface Annexin V and caspase-3 and caspase-9 activities. Further, MCHB colocalizes with mitochondria and disrupts their electrochemical potential. Thus, killing by CNOB involves MCHB, which likely induces apoptosis through the mitochondrial pathway. An attractive feature of the CNOB/ChrR6 regimen is that its toxic product, MCHB, is fluorescent. This feature proved helpful in in vitro studies because simple fluorescence measurements provided information on the kinetics of CNOB activation within the cells, MCHB killing mechanism, its generally efficient bystander effect in cells and cell spheroids, and its biodistribution. The emission wavelength of MCHB also permitted its visualization in live animals, allowing noninvasive qualitative imaging of MCHB in mice and the tumor microenvironment. This feature may simplify exploration of barriers to the penetration of MCHB in tumors and their amelioration. [Mol Cancer Ther 2009;8(2):333–41]

Strategies to achieve systemic delivery of therapeutic cells and microbes to tumors

In order to more effectively treat cancer, targeted delivery of therapeutic agents will be needed. The creation of delivery vehicles capable of locating and entering tumors before delivering a therapeutic payload will, therefore, enable the design of more beneficial and less toxic treatment platforms. Although nanoparticles, microbubbles and liposomes may also partially address these issues, the use of biological agents as delivery vehicles presently holds much promise. Through the hijacking of natural pathogen or cell trafficking pathways it is possible to actively target such agents to the tumor; they are then capable of selective replication (multiplying their therapeutic potential) and may be directly cytolytic themselves and/or may be utilized to deliver therapeutic genes. These agents, such as oncolytic viruses, attenuated bacteria and eukaryotic cells (cellular immunotherapeutics and progenitor and stem cells) will be discussed along with the mechanisms employed to deliver them systemically to tumors, including disseminated disease and micrometsastases.

Role of nitric oxide in Salmonella typhimurium-mediated cancer cell killing

BackgroundBacterial targeting of tumours is an important anti-cancer strategy. We previously showed that strain SL7838 of Salmonella typhimurium targets and kills cancer cells. Whether NO generation by the bacteria has a role in SL7838 lethality to cancer cells is explored. This bacterium has the mechanism for generating NO, but also for decomposing it.MethodsMechanism underlying Salmonella typhimurium tumour therapy was investigated through in vitro and in vivo studies. NO measurements were conducted either by chemical assays (in vitro) or using Biosensors (in vivo). Cancer cells cytotoxic assay were done by using MTS. Bacterial cell survival and tumour burden were determined using molecular imaging techniques.ResultsSL7838 generated nitric oxide (NO) in anaerobic cell suspensions, inside infected cancer cells in vitro and in implanted 4T1 tumours in live mice, the last, as measured using microsensors. Thus, under these conditions, the NO generating pathway is more active than the decomposition pathway. The latter was eliminated, in strain SL7842, by the deletion of hmp- and norV genes, making SL7842 more proficient at generating NO than SL7838. SL7842 killed cancer cells more effectively than SL7838 in vitro, and this was dependent on nitrate availability. This strain was also ca. 100% more effective in treating implanted 4T1 mouse tumours than SL7838.ConclusionsNO generation capability is important in the killing of cancer cells by Salmonella strains.

A General Method for Conditional Regulation of Protein Stability in Living Animals

The ability to rapidly and reversibly perturb protein levels in living animals is a powerful tool for researchersto determine protein function in complex systems. We recently designed a small protein domain based onthe 12-kDa FKBP (FK506 binding protein) that can be fused at either the carboxyl or amino terminus of aprotein of interest. This destabilization domain (DD) confers instability to fusion protein partners, allowingtargeted degradation of the protein of interest. A small molecule called Shield-1 binds to the DD and protectsthe fusion protein from degradation. Small-molecule-mediated post-translational regulation of proteinstability affords this system rapid, reversible, and tunable control of protein levels and functions in a varietyof model systems. Theoretically, a number of transgene delivery methods (e.g., viral, liposomal, or stem cell)can be used for the analysis of a DD fusion protein in an animal model. This protocol uses tumor xenograftsin mice as one such mechanism for delivering the fusion protein and presents a method for deliveringShield-1 to regulate the fusion proteins in vivo.