Investigation of atovaquone-induced spatial changes in tumour hypoxia assessed by hypoxia PET/CT in non-small cell lung cancer patients

Background Tumour hypoxia promotes an aggressive tumour phenotype and enhances resistance to anticancer treatments. Following the recent observation that the mitochondrial inhibitor atovaquone increases tumour oxygenation in NSCLC, we sought to assess whether atovaquone affects tumour subregions differently depending on their level of hypoxia. Methods Patients with resectable NSCLC participated in the ATOM trial (NCT02628080). Cohort 1 (n = 15) received atovaquone treatment, whilst cohort 2 (n = 15) did not. Hypoxia-related metrics, including change in mean tumour-to-blood ratio, tumour hypoxic volume, and fraction of hypoxic voxels, were assessed using hypoxia PET imaging. Tumours were divided into four subregions or distance categories: edge, outer, inner, and centre, using MATLAB. Results Atovaquone-induced reduction in tumour hypoxia mostly occurred in the inner and outer tumour subregions, and to a lesser extent in the centre subregion. Atovaquone did not seem to act in the edge subregion, which was the only tumour subregion that was non-hypoxic at baseline. Notably, the most intensely hypoxic tumour voxels, and therefore the most radiobiologically resistant areas, were subject to the most pronounced decrease in hypoxia in the different subregions. Conclusions This study provides insights into the action of atovaquone in tumour subregions that help to better understand its role as a novel tumour radiosensitiser. Trial registration: ClinicalTrials.gov, NCT0262808. Registered 11th December 2015, https://clinicaltrials.gov/ct2/show/NCT02628080

Development of bacterial nitroreductase enzymes for noninvasive imaging in cancer gene therapy

<p>There is strong interest in developing novel targeted cancer therapies. It has been known for over a century that certain viruses and bacteria can preferentially infect and lyse cancerous cells. Clinical utility has lagged behind the initial promise of the idea; however three therapeutic agents from the oncolytic virus field are currently in Phase IIB/Phase III clinical trials. The development path of such therapies would be substantially smoothed by an ability to nonin vasively monitor the ir location in the patient’s body post-administration. This would allay fears that viral/bacterial distribution may not be confined to the tumour and provide real time information on vector localisation and replication. This could be achieved by positron emission tomography (PET) scanning if the vector expressed a reporter protein which could activate a PET suitable imaging agent. Furthermore the potency of such therapies could be increased by if this reporter protein could also act therapeutically by converting a systemically delivered benign prodrug into a potent chemotherapeutic – thus targeting the toxicity of the prodrug specifically to cancerous cells. A promising enzyme/prodrug combination is the use of bacterial nitroreductase (NTR) enzymes to activate DNA damaging prodrugs, such as the dinitrobenzamides CB1954 and PR-104A. This thesis presents work aimed at developing the ability to noninvasively image bacterial NTR expression so that these enzymes can act as both therapeutic and reporter proteins. The primary focus of this study was to achieve this by repurposing pre-existing 2-nitroimidazole (NI) PET imaging agents, originally developed for imaging tumour hypoxia. Microplate based screening strategies were developed to enable detection of 2-NI bioreductive activation by different bacterial NTRs over-expressed heterologously in Escherichia coli, and these technologies were used to screen a 58-membered library of nitroreductase candidates. Although the most widely studied NTR for enzyme/prodrug therapy - NfsB from E. coli - was found to lack activity with 2-NI substrates, numerous NTRs from the NfsA family were able to metabolise these molecules to the cell entrapped form required for PET imaging. Following this discovery, a directed evolution study was conducted to improve the native activity of the enzyme NfsA from E. coli. In this study targeted mutagenesis of active site residues was carried out, resulting in identification of several NfsA multi-site mutants that were substantially improved in their ability to activate a range of 2-NI imaging agents. In addition to repurposing existing PET probes, this work sought to identify and engineer NTRs for efficient activation of a next - generation PET probe that is designed to be substantially less responsive to hypoxia and hence give a cleaner signal for NTR imaging (i.e. low to no background resulting from tumour hypoxia). SN 33623, a novel 5-NI analogue of the existing 2-NI PET probe EF5, was designed and synthesised by our University of Auckland collaborators. It was found that this novel probe was not only activated by NfsA enzymes, but also by a subset of NfsB enzymes. Although this subset did not include E. coli NfsB, sequence alignment and site-directed mutagenesis were used to identify two key mutations that can be introduced into E. coli NfsB (as well as engineered variants thereof) to confer high levels of SN 33623 activity. Finally work was carried out, as part of a wider collaborative project, to generate NfsA mutants that retained the ability to metabolise 2-NI imaging agents while also showing increased activation of the nitroaromatic prodrug PR-104A. Ongoing evaluation of these enzymes will include assessment of their therapeutic effect in preclinical models and their ability to be noninvasively imaged (by microPET) when expressed from the tumour targeting bacterial strain Clostridium sporogenes.</p>

Development of bacterial nitroreductase enzymes for noninvasive imaging in cancer gene therapy

<p>There is strong interest in developing novel targeted cancer therapies. It has been known for over a century that certain viruses and bacteria can preferentially infect and lyse cancerous cells. Clinical utility has lagged behind the initial promise of the idea; however three therapeutic agents from the oncolytic virus field are currently in Phase IIB/Phase III clinical trials. The development path of such therapies would be substantially smoothed by an ability to nonin vasively monitor the ir location in the patient’s body post-administration. This would allay fears that viral/bacterial distribution may not be confined to the tumour and provide real time information on vector localisation and replication. This could be achieved by positron emission tomography (PET) scanning if the vector expressed a reporter protein which could activate a PET suitable imaging agent. Furthermore the potency of such therapies could be increased by if this reporter protein could also act therapeutically by converting a systemically delivered benign prodrug into a potent chemotherapeutic – thus targeting the toxicity of the prodrug specifically to cancerous cells. A promising enzyme/prodrug combination is the use of bacterial nitroreductase (NTR) enzymes to activate DNA damaging prodrugs, such as the dinitrobenzamides CB1954 and PR-104A. This thesis presents work aimed at developing the ability to noninvasively image bacterial NTR expression so that these enzymes can act as both therapeutic and reporter proteins. The primary focus of this study was to achieve this by repurposing pre-existing 2-nitroimidazole (NI) PET imaging agents, originally developed for imaging tumour hypoxia. Microplate based screening strategies were developed to enable detection of 2-NI bioreductive activation by different bacterial NTRs over-expressed heterologously in Escherichia coli, and these technologies were used to screen a 58-membered library of nitroreductase candidates. Although the most widely studied NTR for enzyme/prodrug therapy - NfsB from E. coli - was found to lack activity with 2-NI substrates, numerous NTRs from the NfsA family were able to metabolise these molecules to the cell entrapped form required for PET imaging. Following this discovery, a directed evolution study was conducted to improve the native activity of the enzyme NfsA from E. coli. In this study targeted mutagenesis of active site residues was carried out, resulting in identification of several NfsA multi-site mutants that were substantially improved in their ability to activate a range of 2-NI imaging agents. In addition to repurposing existing PET probes, this work sought to identify and engineer NTRs for efficient activation of a next - generation PET probe that is designed to be substantially less responsive to hypoxia and hence give a cleaner signal for NTR imaging (i.e. low to no background resulting from tumour hypoxia). SN 33623, a novel 5-NI analogue of the existing 2-NI PET probe EF5, was designed and synthesised by our University of Auckland collaborators. It was found that this novel probe was not only activated by NfsA enzymes, but also by a subset of NfsB enzymes. Although this subset did not include E. coli NfsB, sequence alignment and site-directed mutagenesis were used to identify two key mutations that can be introduced into E. coli NfsB (as well as engineered variants thereof) to confer high levels of SN 33623 activity. Finally work was carried out, as part of a wider collaborative project, to generate NfsA mutants that retained the ability to metabolise 2-NI imaging agents while also showing increased activation of the nitroaromatic prodrug PR-104A. Ongoing evaluation of these enzymes will include assessment of their therapeutic effect in preclinical models and their ability to be noninvasively imaged (by microPET) when expressed from the tumour targeting bacterial strain Clostridium sporogenes.</p>

Targeting Hypoxia: Revival of Old Remedies

Tumour hypoxia is significantly correlated with patient survival and treatment outcomes. At the molecular level, hypoxia is a major driving factor for tumour progression and aggressiveness. Despite the accumulative scientific and clinical efforts to target hypoxia, there is still a need to find specific treatments for tumour hypoxia. In this review, we discuss a variety of approaches to alter the low oxygen tumour microenvironment or hypoxia pathways including carbogen breathing, hyperthermia, hypoxia-activated prodrugs, tumour metabolism and hypoxia-inducible factor (HIF) inhibitors. The recent advances in technology and biological understanding reveal the importance of revisiting old therapeutic regimens and repurposing their uses clinically.

Perfluorocarbons therapeutics in modern cancer nanotechnology for hypoxia-induced anti-tumor therapy

: With an estimated failure rate of about 90%, immunotherapies that are intended for the treatment of solid tumors have caused an anomalous rise in the mortality rate over the past decades. It is apparent that resistance towards such therapies primarily occurs due to elevated levels of HIF-1 (Hypoxia-induced factor) in tumor cells, which are caused by disrupted microcirculation and diffusion mechanisms. With the advent of nanotechnology, several innovative advances were brought to the fore; and, one such promising direction is the use of perfluorocarbon nanoparticles in the management of solid tumors. Perfluorocarbon nanoparticles enhance the response of hypoxia-based agents (HBAs) within the tumor cells and have been found to augment the entry of HBAs into the tumor micro-environment. The heightened penetration of HBAs causes chronic hypoxia, thus aiding in the process of cell quiescence. In addition, this technology has also been applied in photodynamic therapy, where oxygen self-enriched photosensitizers loaded perfluorocarbon nanoparticles are employed. The resulting processes initiate a cascade, depleting tumour oxygen and turning it into a reactive oxygen species eventually to destroy the tumour cell. This review elaborates on the multiple applications of nanotechnology based perfluorocarbon formulations that are being currently employed in the treatment of tumour hypoxia.

Targeted and Non-Targeted Mechanisms for Killing Hypoxic Tumour Cells—Are There New Avenues for Treatment?

Purpose: A major issue in radiotherapy is the relative resistance of hypoxic cells to radiation. Historic approaches to this problem include the use of oxygen mimetic compounds to sensitize tumour cells, which were unsuccessful. This review looks at modern approaches aimed at increasing the efficacy of targeting and radiosensitizing hypoxic tumour microenvironments relative to normal tissues and asks the question of whether non-targeted effects in radiobiology may provide a new “target”. Novel techniques involve the integration of recent technological advancements such as nanotechnology, cell manipulation, and medical imaging. Particularly, the major areas of research discussed in this review include tumour hypoxia imaging through PET imaging to guide carbogen breathing, gold nanoparticles, macrophage-mediated drug delivery systems used for hypoxia-activate prodrugs, and autophagy inhibitors. Furthermore, this review outlines several features of these methods, including the mechanisms of action to induce radiosensitization, the increased accuracy in targeting hypoxic tumour microenvironments relative to normal tissue, preclinical/clinical trials, and future considerations. Conclusions: This review suggests that the four novel tumour hypoxia therapeutics demonstrate compelling evidence that these techniques can serve as powerful tools to increase targeting efficacy and radiosensitizing hypoxic tumour microenvironments relative to normal tissue. Each technique uses a different way to manipulate the therapeutic ratio, which we have labelled “oxygenate, target, use, and digest”. In addition, by focusing on emerging non-targeted and out-of-field effects, new umbrella targets are identified, which instead of sensitizing hypoxic cells, seek to reduce the radiosensitivity of normal tissues.

Tumour Hypoxia-Mediated Immunosuppression: Mechanisms and Therapeutic Approaches to Improve Cancer Immunotherapy

The magnitude of the host immune response can be regulated by either stimulatory or inhibitory immune checkpoint molecules. Receptor-ligand binding between inhibitory molecules is often exploited by tumours to suppress anti-tumour immune responses. Immune checkpoint inhibitors that block these inhibitory interactions can relieve T-cells from negative regulation, and have yielded remarkable activity in the clinic. Despite this success, clinical data reveal that durable responses are limited to a minority of patients and malignancies, indicating the presence of underlying resistance mechanisms. Accumulating evidence suggests that tumour hypoxia, a pervasive feature of many solid cancers, is a critical phenomenon involved in suppressing the anti-tumour immune response generated by checkpoint inhibitors. In this review, we discuss the mechanisms associated with hypoxia-mediate immunosuppression and focus on modulating tumour hypoxia as an approach to improve immunotherapy responsiveness.