Description: An in vivo photomicronucleus test (MNT) using rat skin, the target organ for photoirritancy and carcinogenicity, was recently described. The assay was evaluated using fluoroquinolone (FQ) antibiotics with varying degrees of phototoxic potency (i.e. sparflocacin [SPFX], lomefloxacin [LOFX], ciprofloxacin [CIFX], levofloxacin [LEFX], gemifloxacin [GEFX] and gatifloxacin [GAFX]) using a solar simulator producing both UVA and UVB (ratio 23:1). Experiments were performed at The Netherlands Organisation for Applied Scientific Research (TNO) and GlaxoSmithKline (GSK) to investigate interlaboratory variability, including evaluation of phototoxicity (clinical signs), micronucleus induction and histopathology. The potency of micronuclei (MN) formation in rat skin induced by the FQs was SPFX = LOFX 〉 CIFX = LEFX, however, MN induction was only statistically significant for SPFX and LOFX. In both laboratories, GEFX and GAFX did not increase the MN frequencies compared to the irradiated vehicle control. Signs of phototoxicity, including clinical and histopathological changes, were observed with SPFX and LOFX to a similar degree as the positive control, 8-methoxypsoralen. In addition, there were some clinical signs of phototoxicity seen with CIFX, LEFX, GEFX and GAFX, but not always in both laboratories for CIFX, GEFX and GAFX and when observed, these were considered only mild. Of these, only LEFX also showed histopathological changes. In all studies, photogenotoxic potency correlated with photocarcinogenic potential and moreover, photogenotoxicity was not observed in the absence of phototoxicity. The results of the TNO/GSK study indicate that the in vivo rat skin photoMNT may be a promising tool for detection of photoclastogencity and photoirritancy in the skin/eye in the same animal. Given the association between the MNT and cancer, the skin photoMNT may also provide a promising tool for the early detection of photocarcinogenesis and help bridge the gap in the existing photosafety testing paradigm.

Description: Antibiotics like fluoroquinolones (FQs) that target bacterial type II topoisomerases pose a potential genotoxic risk due to interactions with mammalian topoisomerase II (TOPO II) counterparts. Inhibition of TOPO II can lead to the generation of clastogenic DNA double-strand breaks (DSBs) that can in turn manifest in mutagenesis. Thus, methods that allow early identification of drugs that present the greatest hazard are warranted. A rapid, medium-throughput and predictive genotoxicity screen that can be applied to bacterial type II topoisomerase inhibitors is described herein. Maximal induction of the DSB biomarker serine 139-phosphorylated histone H2AX (H2AX) in L5178Y cells was quantified via flow cytometry and correlated with data derived from the mouse lymphoma screen (MLS), a default assay used to rank genotoxic potential. When applied to a class of novel bacterial type II topoisomerase inhibitors (NBTIs) in lead-optimisation, maximal H2AX induction 〉1.4-fold (relative to controls) identified 22/27 NBTIs that induced 〉6-fold relative mutation frequency (MF) in MLS. Moreover, response signatures comprising of H2AX induction and G 2 M cell cycle arrest elucidated using this approach suggested that these NBTIs, primarily of the H class, operated via a TOPO II poison-like mechanism of action (MoA) similar to FQs. NBTIs that induced ≤6-fold relative MF, which were mainly A class-derived, had less impact on H2AX (≤1.4-fold) and also evoked G 1 arrest, indicating that their cytotoxic effects were likely mediated through a non-poison MoA. Concordance between assays was 86% (54/63) when 1.4- and 6-fold ‘cut offs’ were applied. These findings were corroborated through inspection of human TOPO IIα IC 50 data as NBTIs exhibiting equivalent inhibitory capacities had differing genotoxic potencies. Deployed in an early screening capacity, the H2AX by flow assay coupled with structure–activity relationship evaluation can provide insight into MoA and impact medicinal chemistry efforts, ultimately leading to the production of inherently safer molecules.

Description: The ICH S6(R1) recommendations on safety evaluation of biotherapeutics have led to uncertainty in determining what would constitute a cause for concern that would require genotoxicity testing. A Health and Environmental Sciences Institute’s Genetic Toxicology Technical Committee Workgroup was formed to review the current practice of genotoxicity assessment of peptide/protein-related biotherapeutics. There are a number of properties of peptide/protein-related biotherapeutics that distinguish such products from traditional ‘small molecule’ drugs and need to be taken into consideration when assessing whether genotoxicity testing may be warranted and if so, how to do it appropriately. Case examples were provided by participating companies and decision trees were elaborated to determine whether and when genotoxicity evaluation is needed for peptides containing natural amino acids, non-natural amino acids and other chemical entities and for unconjugated and conjugated proteins. From a scientific point of view, there is no reason for testing peptides containing exclusively natural amino acids irrespective of the manufacturing process. If non-natural amino acids, organic linkers and other non-linker chemical components have already been tested for genotoxicity, there is no need to re-evaluate them when used in different peptide/protein-related biotherapeutics. Unless the peptides have been modified to be able to enter the cells, it is generally more appropriate to evaluate the peptides containing the non-natural amino acids and other non-linker chemical moieties in vivo where the cleavage products can be formed. For linkers, it is important to determine if exposure to reactive forms are likely to occur and from which origin. When the linkers are anticipated to be potential mutagenic impurities they should be evaluated according to ICH M7. If linkers are expected to be catabolic products, it is recommended to test the entire conjugate in vivo , as this would ensure that the relevant ‘free’ linker forms stemming from in vivo catabolism are tested.

Description: Background: The androgen receptor (AR) is a major drug target in prostate cancer (PCa). We profiled the AR-regulated kinome to identify clinically relevant and druggable effectors of AR signaling. Methods: Using genome-wide approaches, we interrogated all AR regulated kinases. Among these, choline kinase alpha (CHKA) expression was evaluated in benign (n = 195), prostatic intraepithelial neoplasia (PIN) (n = 153) and prostate cancer (PCa) lesions (n = 359). We interrogated how CHKA regulates AR signaling using biochemical assays and investigated androgen regulation of CHKA expression in men with PCa, both untreated (n = 20) and treated with an androgen biosynthesis inhibitor degarelix (n = 27). We studied the effect of CHKA inhibition on the PCa transcriptome using RNA sequencing and tested the effect of CHKA inhibition on cell growth, clonogenic survival and invasion. Tumor xenografts (n = 6 per group) were generated in mice using genetically engineered prostate cancer cells with inducible CHKA knockdown. Data were analyzed with 2 tests, Cox regression analysis, and Kaplan-Meier methods. All statistical tests were two-sided. Results: CHKA expression was shown to be androgen regulated in cell lines, xenografts, and human tissue (log fold change from 6.75 to 6.59, P = .002) and was positively associated with tumor stage. CHKA binds directly to the ligand-binding domain (LBD) of AR, enhancing its stability. As such, CHKA is the first kinase identified as an AR chaperone. Inhibition of CHKA repressed the AR transcriptional program including pathways enriched for regulation of protein folding, decreased AR protein levels, and inhibited the growth of PCa cell lines, human PCa explants, and tumor xenografts. Conclusions: CHKA can act as an AR chaperone, providing, to our knowledge, the first evidence for kinases as molecular chaperones, making CHKA both a marker of tumor progression and a potential therapeutic target for PCa.

Description: BACKGROUND Alterations in cardiovascular structure and function have been shown to precede the finding of elevated blood pressure. METHODS This study is part of the Hypertension Genetic Epidemiologic Network (HyperGEN) in which genetic and environmental determinants of hypertension were investigated in 5 geographical field centers. All nonhypertensive offspring (n = 1,035) were included from the entire HyperGEN study population that consists of 2,225 hypertensive patients and 1,380 nonhypertensive patients who had adequate echocardiographic left ventricular (LV) mass measurements. Participants were compared by self-declared race (African American and white). RESULTS Nonhypertensive African American offspring were younger (aged 31 years vs. 38 years), more likely to be female, and had a higher body mass index (BMI) and higher systolic blood pressure (SBP) than their white counterparts. After adjusting for age, sex, SBP, pulse pressure (PP), BMI, diabetes status, and family effects, we observed statistically significant and potentially pathophysiological differences (all with P ≤ 0.001) with greater LV mass/height, relative wall thickness, and posterior wall thickness and with lesser midwall shortening, PP/stroke volume, and (PP/stroke volume)/fat-free body mass. CONCLUSION This study shows that ethnic differences in hemodynamic and echocardiographic profiles exist in a large, population-based cohort of nonhypertensive offspring of hypertensive parents.

Description: Somatic variant analysis of a tumour sample and its matched normal has been widely used in cancer research to distinguish germline polymorphisms from somatic mutations. However, due to the extensive intratumour heterogeneity of cancer, sequencing data from a single tumour sample may greatly underestimate the overall mutational landscape. In recent studies, multiple spatially or temporally separated tumour samples from the same patient were sequenced to identify the regional distribution of somatic mutations and study intratumour heterogeneity. There are a number of tools to perform somatic variant calling from matched tumour-normal next-generation sequencing (NGS) data; however none of these allow joint analysis of multiple same-patient samples. We discuss the benefits and challenges of multisample somatic variant calling and present multiSNV, a software package for calling single nucleotide variants (SNVs) using NGS data from multiple same-patient samples. Instead of performing multiple pairwise analyses of a single tumour sample and a matched normal, multiSNV jointly considers all available samples under a Bayesian framework to increase sensitivity of calling shared SNVs. By leveraging information from all available samples, multiSNV is able to detect rare mutations with variant allele frequencies down to 3% from whole-exome sequencing experiments.