HDAC inhibitors increase NRF2-signaling in tumour cells and blunt the efficacy of co-adminstered cytotoxic agents

The NRF2 signalling cascade provides a primary response against electrophilic chemicals and oxidative stress. The activation of NRF2-signaling is anticipated to have adverse clinical consequences; NRF2 is activated in a number of cancers and, additionally, its pharmacological activation by one compound can reduce the toxicity or efficiency of a second agent administered concomitantly. In this work, we have analysed systematically the ability of 152 research, pre-clinical or clinically used drugs to induce an NRF2 response using the MCF7-AREc32 NRF2 reporter. Ten percent of the tested drugs induced an NRF2 response. The NRF2 activators were not restricted to classical cytotoxic alkylating agents but also included a number of emerging anticancer drugs, including an IGF1-R inhibitor (NVP-AEW541), a PIM-1 kinase inhibitor (Pim1 inhibitor 2), a PLK1 inhibitor (BI 2536) and most strikingly seven of nine tested HDAC inhibitors. These findings were further confirmed by demonstrating NRF2-dependent induction of endogenous AKR genes, biomarkers of NRF2 activity. The ability of HDAC inhibitors to stimulate NRF2-signalling did not diminish their own potency as antitumour agents. However, when used to pre-treat cells, they did reduce the efficacy of acrolein. Taken together, our data suggest that the ability of drugs to stimulate NRF2 activity is common and should be investigated as part of the drug-development process

In vivo stress reporters as early biomarkers of the cellular changes associated with progeria

Age‐related diseases account for a high proportion of the total global burden of disease. Despite recent advances in understanding their molecular basis, there is a lack of suitable early biomarkers to test selected compounds and accelerate their translation to clinical trials. We have investigated the utility of in vivo stress reporter systems as surrogate early biomarkers of the degenerative disease progression. We hypothesized that cellular stress observed in models of human degenerative disease preceded overt cellular damage and at the same time will identify potential cytoprotective pathways. To test this hypothesis, we generated novel accelerated ageing (progeria) reporter mice by crossing the LmnaG609G mice into our oxidative stress/inflammation (Hmox1) and DNA damage (p21) stress reporter models. Histological analysis of reporter expression demonstrated a time‐dependent and tissue‐specific activation of the reporters in tissues directly associated with Progeria, including smooth muscle cells, the vasculature and gastrointestinal tract. Importantly, reporter expression was detected prior to any perceptible deleterious phenotype. Reporter expression can therefore be used as an early marker of progeria pathogenesis and to test therapeutic interventions. This work also demonstrates the potential to use stress reporter approaches to study and find new treatments for other degenerative diseases

Improving the predictive power of xenograft and syngeneic anti-tumour studies using mice humanised for pathways of drug metabolism

Drug development is an expensive and time-consuming process, with only a small fraction of drugs gaining regulatory approval from the often many thousands of candidates identified during target validation. Once a lead compound has been identified and optimised, they are subject to intensive pre-clinical research to determine their pharmacodynamic, pharmacokinetic and toxicological properties, procedures which inevitably involve significant numbers of animals - mainly mice and rats, but also dogs and monkeys in much smaller numbers and for specific types of drug candidates. Many compounds that emerge from this process, having been shown to be safe and efficacious in pre-clinical studies, subsequently fail to replicate this outcome in clinical trials, therefore wasting time, money and, most importantly, animals. Due to high rates of metabolism and a differing spectrum of metabolites (some pharmacologically active) in rodents, species differences in drug metabolism can be a major impediment to drug discovery programmes and confound the extrapolation of animal data to humans. To circumvent this, we have developed a complex transgenic mouse model – 8HUM - which faithfully replicates human Phase I drug metabolism (and its regulation), and which will generate more human-relevant data from fewer animals in a pre-clinical setting and reduce attrition in the clinic. One key area for the pre-clinical application of animals in an oncology setting – almost exclusively mice - is their use in anti-tumour studies. We now further demonstrate the utility of the 8HUM mouse using a murine melanoma cell line as a syngeneic tumour and also present an immunodeficient version 8HUM_Rag2 -/- - for use in xenograft studies. These models will be of significant benefit not only to Pharma for pre-clinical drug development work, but also throughout the drug efficacy, toxicology, pharmacology, and drug metabolism communities, where fewer animals will be needed to generate more human-relevant data

Potential of in vivo stress reporter models to reduce animal use and provide mechanistic insights in toxicity studies

Chemical risk assessment ensures protection from the toxic effects of drugs and manmade chemicals. To comply with regulatory guidance, studies in complex organisms are required, as well as mechanistic studies to establish the relevance of any toxicities observed to man. Although in vitro toxicity models are improving, in vivo studies remain central to this process. Such studies are invariably time-consuming and often involve large numbers of animals. New regulatory frameworks recommend the implementation of “smart” in vivo approaches to toxicity testing that can effectively assess safety for humans and comply with societal expectations for reduction in animal use. A major obstacle in reducing the animals required is the time-consuming and complexity of the pathological endpoints used as markers of toxicity. Such endpoints are prone to inter-animal variability, subjectivity and require harmonisation between testing sites. As a consequence, large numbers of animals per experimental group are required. To address this issue, we propose the implementation of sophisticated stress response reporter mice that we have developed. These reporter models provide early biomarkers of toxic potential in a highly reproducible manner at single-cell resolution, which can also be measured non-invasively and have been extensively validated in academic research as early biomarkers of stress responses for a wide range of chemicals at human-relevant exposures. In this report, we describe a new and previously generated models in our lab, provide the methodology required for their use and discuss how they have been used to inform on toxic risk. We propose our in vivo approach is more informative (refinement) and reduces the animal use (reduction) compared to traditional toxicity testing. These models could be incorporated into tiered toxicity testing and used in combination with in vitro assays to generate quantitative adverse outcome pathways and inform on toxic potential