Model for MLL translocations in therapy-related leukemia involving topoisomerase IIß-mediated DNA strand breaks and gene proximity.

Topoisomerase poisons such as the epipodophyllotoxin etoposide are widely used effective cytotoxic anticancer agents. However, they are associated with the development of therapy-related acute myeloid leukemias (t-AMLs), which display characteristic balanced chromosome translocations, most often involving the mixed lineage leukemia (MLL) locus at 11q23. MLL translocation breakpoints in t-AMLs cluster in a DNase I hypersensitive region, which possesses cryptic promoter activity, implicating transcription as well as topoisomerase II activity in the translocation mechanism. We find that 2-3% of MLL alleles undergoing transcription do so in close proximity to one of its recurrent translocation partner genes, AF9 or AF4, consistent with their sharing transcription factories. We show that most etoposide-induced chromosome breaks in the MLL locus and the overall genotoxicity of etoposide are dependent on topoisomerase IIß, but that topoisomerase IIα and -ß occupancy and etoposide-induced DNA cleavage data suggest factors other than local topoisomerase II concentration determine specific clustering of MLL translocation breakpoints in t-AML. We propose a model where DNA double-strand breaks (DSBs) introduced by topoisomerase IIß into pairs of genes undergoing transcription within a common transcription factory become stabilized by antitopoisomerase II drugs such as etoposide, providing the opportunity for illegitimate end joining and translocation.

The role of topoisomerase II beta on breakage and proximity of RUNX1 to partner alleles RUNX1T1 and EVI1.

Rearrangements involving the RUNX1 gene account for approximately 15% of balanced translocations in therapy-related acute myeloid leukemia (t-AML) patients and are one of the most common genetic abnormalities observed in t-AML. Drugs targeting the topoisomerase II (TOP2) enzyme are implicated in t-AML; however, the mechanism is not well understood and to date a single RUNX1-RUNX1T1 t-AML breakpoint junction sequence has been published. Here we report an additional five breakpoint junction sequences from t-AML patients with the RUNX1- RUNX1T1 translocation. Using a leukemia cell line model, we show that TOP2 beta (TOP2B) is required for induction of RUNX1 chromosomal breaks by the TOP2 poison etoposide and that, while TOP2 alpha (TOP2A) and TOP2B proteins are both present on RUNX1 and RUNX1T1 chromatin, only the TOP2B enrichment reached significance following etoposide exposure at a region on RUNX1 where translocations occur. Furthermore, we demonstrate that TOP2B influences the separation between RUNX1 and two translocation partners (RUNX1T1 and EVI) in the nucleus of lymphoid cells. Specifically, we identified a TOP2B-dependent increase in the number of nuclei displaying juxtaposed RUNX1 and RUNX1T1 loci following etoposide treatment.

Identity leadership and adherence to COVID-19 safety guidance in hospital settings.

COVID-19 presents unique and complex challenges to the Scottish National Health Service (NHS). As COVID-19 preventative measures are effective at reducing disease spread, promoting staff adherence in high-risk workplaces is vital. The present research explored the role of identity leadership on (a) staff's appraisals of leadership and (b) staff's adherence to and attitudes towards COVID-19 guidance. Semi-structured interviews (N = 25) were conducted with NHS staff across two Scottish hospitals. Using Reflexive Thematic Analysis, two over-arching themes were generated: leadership presence and approachable leadership who act on group concerns, where both created positive appraisals of leadership and were seen to facilitate adherence. Guidance from present leaders was perceived as both practical and applicable. Approachable leaders were viewed to facilitate information sharing, clarify guidance, and allow staff to raise concerns. Leaders who were seen to act on group concerns provided resources or updated guidance to promote adherence. The present study provides theoretical and practical advancements to (a) expand the known role of identity leadership in promoting safety in workplaces and (b) facilitate routes for adherence to safety guidance beyond COVID-19.

Comprehensive analysis of differentially expressed miRNAs in hepatocellular carcinoma: Prognostic, predictive significance and pathway insights.

Robust prognostic and predictive factors for hepatocellular carcinoma, a leading cause of cancer-related deaths worldwide, have not yet been identified. Previous studies have identified potential HCC determinants such as genetic mutations, epigenetic alterations, and pathway dysregulation. However, the clinical significance of these molecular alterations remains elusive. MicroRNAs are major regulators of protein expression. MiRNA functions are frequently altered in cancer. In this study, we aimed to explore the prognostic value of differentially expressed miRNAs in HCC, to elucidate their associated pathways and their impact on treatment response. To this aim, bioinformatics techniques and clinical dataset analyses were employed to identify differentially expressed miRNAs in HCC compared to normal hepatic tissue. We validated known associations and identified a novel miRNA signature with potential prognostic significance. Our comprehensive analysis identified new miRNA-targeted pathways and showed that some of these protein coding genes predict HCC patients' response to the tyrosine kinase inhibitor sorafenib.

Social identity processes associated with perceived risk at pilot sporting events during COVID-19.

Previous research suggests that shared social identification and expected support from others can reduce the extent to which attendees of mass events perceive that others pose health risks. This study evaluated the social identity processes associated with perceived risk at UK pilot sporting events held during COVID-19, including the government Events Research Programme. An online survey (N = 2029) measured attendee perceptions that other spectators adhered to safety measures, shared social identity with other attendees, expectations that others would provide support, and the perceived risk of germ spread from other attendees. Results indicate that for football attendees, seeing others adhering to COVID-19 safety measures was associated with lower perceived risk and this was partially mediated via increased shared social identity and expected support. However, the sequential mediations were non-significant for rugby and horse racing events. The decreased perceived risk for football and rugby attendees highlights the importance of understanding social identity processes at mass events to increase safety. The non-significant associations between shared social identity and perceived risk and between expected support and perceived risk for both the rugby and the horse racing highlights the need to further research risk perceptions across a range of mass event contexts.

Topoisomerase II Chromatin Immunoprecipitation.

Chromatin immunoprecipitation is a method to isolate a protein of interest coupled to DNA following cross-linking with formaldehyde and to quantify the relative abundance or occupancy of the protein at specific genomic loci. After immunoprecipitation of protein-DNA complexes protein-DNA cross-links are reversed and the DNA is extracted. Various methods exist to identify binding sites and determine relative occupancy of the protein of interest; these include quantitative PCR, probing microarrays or sequencing the isolated DNA (ChIP-seq). This chapter details the method of chromatin immunoprecipitation of TOP2 to the point of DNA extraction from the precipitated protein-DNA complexes.

Genome-wide ChIP-seq analysis of human TOP2B occupancy in MCF7 breast cancer epithelial cells.

We report the whole genome ChIP seq for human TOP2B from MCF7 cells. Using three different peak calling methods, regions of binding were identified in the presence or absence of the nuclear hormone estradiol, as TOP2B has been reported to play a role in ligand-induced transcription. TOP2B peaks were found across the whole genome, 50% of the peaks fell either within a gene or within 5 kb of a transcription start site. TOP2B peaks coincident with gene promoters were less frequently associated with epigenetic features marking active promoters in estradiol treated than in untreated cells. Significantly enriched transcription factor motifs within the DNA sequences underlying the peaks were identified. These included SP1, KLF4, TFAP2A, MYF, REST, CTCF, ESR1 and ESR2. Gene ontology analysis of genes associated with TOP2B peaks found neuronal development terms including axonogenesis and axon guidance were significantly enriched. In the absence of functional TOP2B there are errors in axon guidance in the zebrafish eye. Specific heparin sulphate structures are involved in retinal axon targeting. The glycosaminoglycan biosynthesis-heparin sulphate/heparin pathway is significantly enriched in the TOP2B gene ontology analysis, suggesting changes in this pathway in the absence of TOP2B may cause the axon guidance faults.