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Objective: The UK Soft Drinks Industry Levy (SDIL) (announced in March 2016; implemented in April 2018) aims to incentivise reformulation of soft drinks to reduce added sugar levels. The SDIL has been applauded as a policy success, and it has survived calls from parliamentarians for it to be repealed. We aimed to explore parliamentary reaction to the SDIL following its announcement until two years post-implementation in order to understand how health policy can become established and resilient to opposition., Design: Searches of Hansard for parliamentary debate transcripts that discussed the SDIL retrieved 186 transcripts, with 160 included after screening. Five stages of Applied Thematic Analysis were conducted: familiarisation and creation of initial codebooks; independent second coding; codebook finalisation through team consensus; final coding of the dataset to the complete codebook; and theme finalisation through team consensus., Setting: The United Kingdom Parliament., Participants: N/A., Results: Between the announcement (16/03/2016) - royal assent (26/04/2017), two themes were identified 1: SDIL welcomed cross-party 2: SDIL a good start but not enough. Between royal assent - implementation (5/04/2018), one theme was identified 3: The SDIL worked - what next? The final theme identified from implementation until 16/03/2020 was 4: Moving on from the SDIL ., Conclusions: After the announcement, the SDIL had cross-party support and was recognised to have encouraged reformulation prior to implementation. Lessons for governments indicate that the combination of cross-party support and a policy's documented success in achieving its aim can help cement the resilience of it to opposition and threats of repeal.

The Healthy New Town programme in England set out to 'put health into place' by supporting the design and construction of healthy places to live, including by creating safe environments for active travel. To explore the impact of this approach, this study examined how children and their families experienced school journeys in two contrasting Healthy New Towns in England, one an affluent new town in the early stages of construction and the other more economically deprived and established. We undertook photo-elicitation and go-along interviews with 24 children aged 7-12 years and semi-structured interviews with 17 caregivers. We found that experiences of care were important for children's school travel. In the 'deprived' town, opportunities for children to care and to be cared for were enjoyed, facilitated by routes with limited traffic, pockets of 'nature', and possibilities to encounter meaningful others. For families living in a town under construction, the need to negotiate unfinished travel infrastructure, and a sense of being 'in limbo', was experienced as an absence of care by planners and developers. Interventions to promote children's active travel should consider the role of care-full planning in facilitating walking and cycling journeys., Competing Interests: Declaration of competing interest None of the authors have any conflicts of interest to declare., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: Despite limited genetic and histologic heterogeneity, Ewing sarcoma (EwS) tumor cells are transcriptionally heterogeneous and display varying degrees of mesenchymal lineage specification in vitro. In this study, we investigated if and how transcriptional heterogeneity of EwS cells contributes to heterogeneity of tumor phenotypes in vivo., Experimental Design: Single-cell proteogenomic-sequencing of EwS cell lines was performed and integrated with patient tumor transcriptomic data. Cell subpopulations were isolated by FACS for assessment of gene expression and phenotype. Digital spatial profiling and human whole transcriptome analysis interrogated transcriptomic heterogeneity in EwS xenografts. Tumor cell subpopulations and matrix protein deposition were evaluated in xenografts and patient tumors using multiplex immunofluorescence staining., Results: We identified CD73 as a biomarker of highly mesenchymal EwS cell subpopulations in tumor models and patient biopsies. CD73+ tumor cells displayed distinct transcriptional and phenotypic properties, including selective upregulation of genes that are repressed by EWS::FLI1, and increased migratory potential. CD73+ cells were distinguished in vitro and in vivo by increased expression of matrisomal genes and abundant deposition of extracellular matrix (ECM) proteins. In epithelial-derived malignancies, ECM is largely deposited by cancer-associated fibroblasts (CAF), and we thus labeled CD73+ EwS cells, CAF-like tumor cells. Marked heterogeneity of CD73+ EwS cell frequency and distribution was detected in tumors in situ, and CAF-like tumor cells and associated ECM were observed in peri-necrotic regions and invasive foci., Conclusions: EwS tumor cells can adopt CAF-like properties, and these distinct cell subpopulations contribute to tumor heterogeneity by remodeling the tumor microenvironment. See related commentary by Kuo and Amatruda, p. 5002., (©2023 American Association for Cancer Research.)

Adrenocortical carcinoma (ACC) is a rare cancer in which tissue-specific differentiation is paradoxically associated with dismal outcomes. The differentiated ACC subtype CIMP-high is prevalent, incurable, and routinely fatal. CIMP-high ACC possess abnormal DNA methylation and frequent β-catenin-activating mutations. Here, we demonstrated that ACC differentiation is maintained by a balance between nuclear, tissue-specific β-catenin-containing complexes, and the epigenome. On chromatin, β-catenin bound master adrenal transcription factor SF1 and hijacked the adrenocortical super-enhancer landscape to maintain differentiation in CIMP-high ACC; off chromatin, β-catenin bound histone methyltransferase EZH2. SF1/β-catenin and EZH2/β-catenin complexes present in normal adrenals persisted through all phases of ACC evolution. Pharmacologic EZH2 inhibition in CIMP-high ACC expelled SF1/β-catenin from chromatin and favored EZH2/β-catenin assembly, erasing differentiation and restraining cancer growth in vitro and in vivo. These studies illustrate how tissue-specific programs shape oncogene selection, surreptitiously encoding targetable therapeutic vulnerabilities., Significance: Oncogenic β-catenin can use tissue-specific partners to regulate cellular differentiation programs that can be reversed by epigenetic therapies, identifying epigenetic control of differentiation as a viable target for β-catenin-driven cancers., (©2023 American Association for Cancer Research.)

Background: The COVID-19 pandemic accelerated the interest in implementing mobile health (mHealth) in population-based health studies, but evidence is lacking on engagement and adherence in studies. We conducted a fully remote study for ≥6 months tracking COVID-19 digital biomarkers and symptoms using a smartphone app nested within an existing cohort of adults., Objective: We aimed to investigate participant characteristics associated with initial and sustained engagement in digital biomarker collection from a bespoke smartphone app and if engagement changed over time or because of COVID-19 factors and explore participants' reasons for consenting to the smartphone substudy and experiences related to initial and continued engagement., Methods: Participants in the Fenland COVID-19 study were invited to the app substudy from August 2020 to October 2020 until study closure (April 30, 2021). Participants were asked to complete digital biomarker modules (oxygen saturation, body temperature, and resting heart rate [RHR]) and possible COVID-19 symptoms in the app 3 times per week. Participants manually entered the measurements, except RHR that was measured using the smartphone camera. Engagement was categorized by median weekly frequency of completing the 3 digital biomarker modules (categories: 0, 1-2, and ≥3 times per week). Sociodemographic and health characteristics of those who did or did not consent to the substudy and by engagement category were explored. Semistructured interviews were conducted with 35 participants who were purposively sampled by sex, age, educational attainment, and engagement category, and data were analyzed thematically; 63% (22/35) of the participants consented to the app substudy, and 37% (13/35) of the participants did not consent., Results: A total of 62.61% (2524/4031) of Fenland COVID-19 study participants consented to the app substudy. Of those, 90.21% (2277/2524) completed the app onboarding process. Median time in the app substudy was 34.5 weeks (IQR 34-37) with no change in engagement from 0 to 3 months or 3 to 6 months. Completion rates (≥1 per week) across the study between digital biomarkers were similar (RHR: 56,517/77,664, 72.77%; temperature: 56,742/77,664, 73.06%; oxygen saturation: 57,088/77,664, 73.51%). Older age groups and lower managerial and intermediate occupations were associated with higher engagement, whereas working, being a current smoker, being overweight or obese, and high perceived stress were associated with lower engagement. Continued engagement was facilitated through routine and personal motivation, and poor engagement was caused by user error and app or equipment malfunctions preventing data input. From these results, we developed key recommendations to improve engagement in population-based mHealth studies., Conclusions: This mixed methods study demonstrated both high initial and sustained engagement in a large mHealth COVID-19 study over a ≥6-month period. Being nested in a known cohort study enabled the identification of participant characteristics and factors associated with engagement to inform future applications in population-based health research., (©Kirsten L Rennie, Emma R Lawlor, Arrash Yassaee, Adam Booth, Kate Westgate, Stephen J Sharp, Carina S B Tyrrell, Mert Aral, Nicholas J Wareham. Originally published in the Journal of Medical Internet Research (https://www.jmir.org), 13.01.2023.)

Accumulating evidence shows that despite clonal origins tumors eventually become complex communities comprised of phenotypically distinct cell subpopulations. This heterogeneity arises from both tumor cell intrinsic programs and signals from spatially and temporally dynamic microenvironments. While pediatric cancers usually lack the mutational burden of adult cancers, they still exhibit high levels of cellular heterogeneity that are largely mediated by epigenetic mechanisms. Ewing sarcomas are aggressive bone and soft tissue malignancies with peak incidence in adolescence and the prognosis for patients with relapsed and metastatic disease is dismal. Ewing sarcomas are driven by a single pathognomonic fusion between a FET protein and an ETS family transcription factor, the most common of which is EWS::FLI1. Despite sharing a single driver mutation, Ewing sarcoma cells demonstrate a high degree of transcriptional heterogeneity both between and within tumors. Recent studies have identified differential fusion protein activity as a key source of this heterogeneity which leads to profoundly different cellular phenotypes. Paradoxically, increased invasive and metastatic potential is associated with lower EWS::FLI1 activity. Here, we review what is currently understood about EWS::FLI1 activity, the cell autonomous and tumor microenvironmental factors that regulate it, and the downstream consequences of these activity states on tumor progression. We specifically highlight how transcription factor regulation, signaling pathway modulation, and the extracellular matrix intersect to create a complex network of tumor cell phenotypes. We propose that elucidation of the mechanisms by which these essential elements interact will enable the development of novel therapeutic approaches that are designed to target this complexity and ultimately improve patient outcomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Apfelbaum, Wrenn and Lawlor.)

Purpose: Propagation of Ewing sarcoma requires precise regulation of EWS::FLI1 transcriptional activity. Determining the mechanisms of fusion regulation will advance our understanding of tumor progression. Here we investigated whether HOXD13, a developmental transcription factor that promotes Ewing sarcoma metastatic phenotypes, influences EWS::FLI1 transcriptional activity., Experimental Design: Existing tumor and cell line datasets were used to define EWS::FLI1 binding sites and transcriptional targets. Chromatin immunoprecipitation and CRISPR interference were employed to identify enhancers. CUT&RUN and RNA sequencing defined binding sites and transcriptional targets of HOXD13. Transcriptional states were investigated using bulk and single-cell transcriptomic data from cell lines, patient-derived xenografts, and patient tumors. Mesenchymal phenotypes were assessed by gene set enrichment, flow cytometry, and migration assays., Results: We found that EWS::FLI1 creates a de novo GGAA microsatellite enhancer in a developmentally conserved regulatory region of the HOXD locus. Knockdown of HOXD13 led to widespread changes in expression of developmental gene programs and EWS::FLI1 targets. HOXD13 binding was enriched at established EWS::FLI1 binding sites where it influenced expression of EWS::FLI1-activated genes. More strikingly, HOXD13 bound and activated EWS::FLI1-repressed genes, leading to adoption of mesenchymal and migratory cell states that are normally suppressed by the fusion. Single-cell analysis confirmed that direct transcriptional antagonism between HOXD13-mediated gene activation and EWS::FLI1-dependent gene repression defines the state of Ewing sarcoma cells along a mesenchymal axis., Conclusions: Ewing sarcoma tumors are comprised of tumor cells that exist along a mesenchymal transcriptional continuum. The identity of cells along this continuum is, in large part, determined by the competing activities of EWS::FLI1 and HOXD13. See related commentary by Weiss and Bailey, p. 4360., (©2022 American Association for Cancer Research.)

Primary bone sarcomas, including osteosarcoma (OS) and Ewing sarcoma (ES), are aggressive tumors with peak incidence in childhood and adolescence. The intense standard treatment for these patients consists of combined surgery and/or radiation and maximal doses of chemotherapy; a regimen that has not seen improvement in decades. Like other tumor types, ES and OS are characterized by dysregulated cellular metabolism and a rewiring of metabolic pathways to support the biosynthetic demands of malignant growth. Not only are cancer cells characterized by Warburg metabolism, or aerobic glycolysis, but emerging work has revealed a dependence on amino acid metabolism. Aside from incorporation into proteins, amino acids serve critical functions in redox balance, energy homeostasis, and epigenetic maintenance. In this review, we summarize current studies describing the amino acid metabolic requirements of primary bone sarcomas, focusing on OS and ES, and compare these dependencies in the normal bone and malignant tumor contexts. We also examine insights that can be gleaned from other cancers to better understand differential metabolic susceptibilities between primary and metastatic tumor microenvironments. Lastly, we discuss potential metabolic vulnerabilities that may be exploited therapeutically and provide better-targeted treatments to improve the current standard of care., Competing Interests: CL has received consulting fees from Astellas Pharmaceuticals, Odyssey Therapeutics, and T-Knife Therapeutics, and is an inventor on patents pertaining to Kras regulated metabolic pathways, redox control pathways in pancreatic cancer, and targeting the GOT1-pathway as a therapeutic approach (US Patent No: 2015126580-A1, 05/07/2015; US Patent No: 20190136238, 05/09/2019; International Patent No: WO2013177426-A2, 04/23/2015). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Jiménez, Lawlor and Lyssiotis.)

The advent of dose intensified interval compressed therapy has improved event-free survival for patients with localized Ewing sarcoma (EwS) to 78% at 5 years. However, nearly a quarter of patients with localized tumors and 60-80% of patients with metastatic tumors suffer relapse and die of disease. In addition, those who survive are often left with debilitating late effects. Clinical features aside from stage have proven inadequate to meaningfully classify patients for risk-stratified therapy. Therefore, there is a critical need to develop approaches to risk stratify patients with EwS based on molecular features. Over the past decade, new technology has enabled the study of multiple molecular biomarkers in EwS. Preliminary evidence requiring validation supports copy number changes, and loss of function mutations in tumor suppressor genes as biomarkers of outcome in EwS. Initial studies of circulating tumor DNA demonstrated that diagnostic ctDNA burden and ctDNA clearance during induction are also associated with outcome. In addition, fusion partner should be a pre-requisite for enrollment on EwS clinical trials, and the fusion type and structure require further study to determine prognostic impact. These emerging biomarkers represent a new horizon in our understanding of disease risk and will enable future efforts to develop risk-adapted treatment., (© 2022. The Author(s).)

The eighth Paediatric Strategy Forum focused on multi-targeted kinase inhibitors (mTKIs) in osteosarcoma and Ewing sarcoma. The development of curative, innovative products in these tumours is a high priority and addresses unmet needs in children, adolescents and adults. Despite clinical and investigational use of mTKIs, efficacy in patients with bone tumours has not been definitively demonstrated. Randomised studies, currently being planned or in progress, in front-line and relapse settings will inform the further development of this class of product. It is crucial that these are rapidly initiated to generate robust data to support international collaborative efforts. The experience to date has generally indicated that the safety profile of mTKIs as monotherapy, and in combination with chemotherapy or other targeted therapy, is consistent with that of adults and that toxicity is manageable. Increasing understanding of relevant predictive biomarkers and tumour biology is absolutely critical to further develop this class of products. Biospecimen samples for correlative studies and biomarker development should be shared, and a joint academic-industry consortium created. This would result in an integrated collection of serial tumour tissues and a systematic retrospective and prospective analyses of these samples to ensure robust assessment of biologic effect of mTKIs. To support access for children to benefit from these novel therapies, clinical trials should be designed with sufficient scientific rationale to support regulatory and payer requirements. To achieve this, early dialogue between academia, industry, regulators, and patient advocates is essential. Evaluating feasibility of combination strategies and then undertaking a randomised trial in the same protocol accelerates drug development. Where possible, clinical trials and development should include children, adolescents, and adults less than 40 years. To respond to emerging science, in approximately 12 months, a multi-stakeholder group will meet and review available data to determine future directions and priorities., Competing Interests: Conflcits of interest statement The authors declare the following financial interests/ personal relationships which may be considered as potential competing interests: FB is an employee and stockholder of Ipsen Pharma. JC is an employee of Bayer Healthcare Pharmaceuticals. MC has served as an advisor for Astra-Zeneca, Bayer, BMS, Pfizer, and Servier. SGD has received consulting fees from Amgen, Bayer, and Loxo Oncology and has received travel reimbursement from Roche and Salarius. EG is an employee of Oncoheroes Biosciences. AH is an employee of Blueprint Medicines. KJ has received consulting fees from Bayer and Ipsen and honoraria fromTakeda and Foundation Medicine. JK is an employee of Allarity Therapeutics. SK is an employee of HUTCHMED International Corporation. CO is an employee of Eisai GmbH. ADJP has consulted for Lilly, Norgine and Developmental Therapeutics Consortium Limited and been an advisor for Amgen. All remaining authors have declared no conflicts of interest., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

This paper uses meta-ethnography to synthesise qualitative and ethnographic studies of children's (aged 5-13) experiences of socio-material environments on their school journey. Most of the 21 papers (18 studies) identified from the systematic search were from high-income countries and used self-report qualitative methods. Our synthesis shows children can feel vulnerable, but also negotiate journeys and manage risks, enjoy shared and solitary mobility, and explore their material environments. School journeys offer children a place to learn and develop agency within their socio-material environments. Attending to these wider benefits of school journeys, alongside supporting children to develop active modes attuned to the risks associated with these journeys, could improve the reach and impact of active school travel initiatives., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Glioblastoma (GBM) is an aggressive primary brain cancer, with a 5 year survival of ∼5%. Challenges that hamper GBM therapeutic efficacy include (i) tumor heterogeneity, (ii) treatment resistance, (iii) immunosuppressive tumor microenvironment (TME), and (iv) the blood-brain barrier (BBB). The C-X-C motif chemokine ligand-12/C-X-C motif chemokine receptor-4 (CXCL12/CXCR4) signaling pathway is activated in GBM and is associated with tumor progression. Although the CXCR4 antagonist (AMD3100) has been proposed as an attractive anti-GBM therapeutic target, it has poor pharmacokinetic properties, and unfavorable bioavailability has hampered its clinical implementation. Thus, we developed synthetic protein nanoparticles (SPNPs) coated with the transcytotic peptide iRGD (AMD3100-SPNPs) to target the CXCL2/CXCR4 pathway in GBM via systemic delivery. We showed that AMD3100-SPNPs block CXCL12/CXCR4 signaling in three mouse and human GBM cell cultures in vitro and in a GBM mouse model in vivo. This results in (i) inhibition of GBM proliferation, (ii) reduced infiltration of CXCR4 + monocytic myeloid-derived suppressor cells (M-MDSCs) into the TME, (iii) restoration of BBB integrity, and (iv) induction of immunogenic cell death (ICD), sensitizing the tumor to radiotherapy and leading to anti-GBM immunity. Additionally, we showed that combining AMD3100-SPNPs with radiation led to long-term survival, with ∼60% of GBM tumor-bearing mice remaining tumor free after rechallenging with a second GBM in the contralateral hemisphere. This was due to a sustained anti-GBM immunological memory response that prevented tumor recurrence without additional treatment. In view of the potent ICD induction and reprogrammed tumor microenvironment, this SPNP-mediated strategy has a significant clinical translation applicability.

Introduction: The cost-effectiveness and long-term health impact of behavioural weight management programmes depends on post-treatment weight-loss maintenance. Growing evidence suggests that interventions using acceptance and commitment therapy (ACT) could improve long-term weight management. We developed an ACT-based, guided self-help intervention to support adults who have recently completed a behavioural weight loss programme. This study will assess the feasibility and acceptability of this type of intervention and findings will inform the development of a full-scale trial., Methods and Analysis: This is a pragmatic, randomised, single-blind, parallel group, two-arm, feasibility study with an embedded process evaluation. We will recruit and randomise 60 adults who have recently completed a behavioural weight loss programme to the ACT-based intervention or standard care, using a computer-generated sequence with 2:1 allocation stratified by diabetes status and sex. Baseline and 6-month measurements will be completed using online questionnaires. Qualitative interviews will be conducted with a subsample of participants and coaches about their experiences at 3 (mid-intervention) and 6 (postintervention) months. Feasibility and acceptability of the intervention, and a full-scale trial will be assessed using a number of outcomes, including adherence to, and engagement with the intervention, recruitment and retention rates, proportion of missing data for each outcome measure, participants' experiences of the intervention and study, and coaches' experiences of delivering intervention support. Quantitative and qualitative findings will be integrated and summarised to contribute to the interpretation of the main feasibility evaluation findings. Value of information methods will be used to estimate the decision uncertainty associated with the intervention's cost-effectiveness and determine the value of a definitive trial., Ethics and Dissemination: Ethical approval was received from Cambridge South Research Ethics Committee on 15/03/2021 (21/EE/0024). This protocol (V.2) was approved on 19 April 2021. Findings will be published in peer-reviewed scientific journals and communicated to other stakeholders as appropriate., Trial Registration Number: ISRCTN12685964., Competing Interests: Competing interests: ALA is principal investigator on another publicly funded trial where JW provided the intervention at no cost but has received no personal fees. SG reports grants from Medical Research Council, personal fees from Eli Lilly and personal fees from Janssen, outside this programme of research. AH reports personal fees from Slimming World and the College of Contemporary Health, outside this programme of research. CH reports informal unpaid advice to Thriva., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

Aims/hypothesis: Weight loss is often recommended in the treatment of type 2 diabetes. While evidence has shown that large weight loss may lead to diabetes remission and improvement in cardiovascular risk factors, long-term impacts are unclear. We performed a systematic review of studies of weight loss and other weight changes and incidence of CVD among people with type 2 diabetes., Methods: Observational studies of behavioural (non-surgical and non-pharmaceutical) weight changes and CVD events among adults with type 2 diabetes, and trials of behavioural interventions targeting weight loss, were identified through searches of MEDLINE, EMBASE, Web of Science, CINAHL, and The Cochrane Library (CENTRAL) until 9 July 2019. Included studies reported change in weight and CVD and/or mortality outcomes among adults with type 2 diabetes. We performed a narrative synthesis of observational studies and meta-analysis of trial data., Results: Of 13,227 identified articles, 17 (14 observational studies, three trials) met inclusion criteria. Weight gain (vs no change) was associated with higher hazard of CVD events (HRs [95% CIs] ranged from 1.13 [1.00, 1.29] to 1.63 [1.11, 2.39]) and all-cause mortality (HRs [95% CIs] ranged from 1.26 [1.12, 1.41] to 1.57 [1.33, 1.85]). Unintentional weight loss (vs no change) was associated with higher risks of all-cause mortality, but associations with intentional weight loss were unclear. Behavioural interventions targeting weight loss showed no effect on CVD events (pooled HR [95% CI] 0.95 [0.71, 1.27]; I 2  = 50.1%). Risk of bias was moderate in most studies and was high in three studies, due to potential uncontrolled confounding and method of weight assessment., Conclusions/interpretation: Weight gain is associated with increased risks of CVD and mortality, although there is a lack of data supporting behavioural weight-loss interventions for CVD prevention among adults with type 2 diabetes. Long-term follow-up of behavioural intervention studies is needed to understand effects on CVD and mortality and to inform policy concerning weight management advice and support for people with diabetes. PROSPERO registration CRD42019127304., (© 2021. The Author(s).)

Background: The EWSR1/FLI1 gene fusion is the most common rearrangement leading to cell transformation in Ewing sarcoma (ES). Previous studies have indicated that expression at the cellular level is heterogeneous, and that levels of expression may oscillate, conferring different cellular characteristics. In ES the role of EWSR1/FLI1 in regulating subpopulation dynamics is currently unknown., Methods: We used siRNA to transiently suppress EWSR1/FLI1 expression and followed population dynamics using both single cell expression profiling, CyTOF and functional assays to define characteristics of exponentially growing ES cells and of ES cells in which EWSR1/FLI1 had been downregulated. Novel transcriptional states with distinct features were assigned using random forest feature selection in combination with machine learning. Cells isolated from ES xenografts in immune-deficient mice were interrogated to determine whether characteristics of specific subpopulations of cells in vitro could be identified. Stem-like characteristics were assessed by primary and secondary spheroid formation in vitro, and invasion/motility was determined for each identified subpopulation. Autophagy was determined by expression profiling, cell sorting and immunohistochemical staining., Results: We defined a workflow to study EWSR1/FLI1 driven transcriptional states and phenotypes. We tracked EWSR1/FLI1 dependent proliferative activity over time to discover sources of intra-tumoral diversity. Single-cell RNA profiling was used to compare expression profiles in exponentially growing populations (si-Control) or in two dormant populations (D1, D2) in which EWSR1/FLI1 had been suppressed. Three distinct transcriptional states were uncovered contributing to ES intra-heterogeneity. Our predictive model identified ~1% cells in a dormant-like state and ~ 2-4% cells with stem-like and neural stem-like features in an exponentially proliferating ES cell line and in ES xenografts. Following EWSR1/FLI1 knockdown, cells re-entering the proliferative cycle exhibited greater stem-like properties, whereas for those cells remaining quiescent, FAM134B-dependent dormancy may provide a survival mechanism., Conclusions: We show that time-dependent changes induced by suppression of oncogenic EWSR1/FLI1 expression induces dormancy, with different subpopulation dynamics. Cells re-entering the proliferative cycle show enhanced stem-like characteristics, whereas those remaining dormant for prolonged periods appear to survive through autophagy. Cells with these characteristics identified in exponentially growing cell populations and in tumor xenografts may confer drug resistance and could potentially contribute to metastasis., (© 2021. Springer Nature Switzerland AG.)

Background: The long-term impact and cost-effectiveness of weight management programs depend on posttreatment weight maintenance. There is growing evidence that interventions based on third-wave cognitive behavioral therapy, particularly acceptance and commitment therapy (ACT), could improve long-term weight management; however, these interventions are typically delivered face-to-face by psychologists, which limits the scalability of these types of intervention., Objective: The aim of this study is to use an evidence-, theory-, and person-based approach to develop an ACT-based intervention for weight loss maintenance that uses digital technology and nonspecialist guidance to minimize the resources needed for delivery at scale., Methods: Intervention development was guided by the Medical Research Council framework for the development of complex interventions in health care, Intervention Mapping Protocol, and a person-based approach for enhancing the acceptability and feasibility of interventions. Work was conducted in two phases: phase 1 consisted of collating and analyzing existing and new primary evidence and phase 2 consisted of theoretical modeling and intervention development. Phase 1 included a synthesis of existing evidence on weight loss maintenance from previous research, a systematic review and network meta-analysis of third-wave cognitive behavioral therapy interventions for weight management, a qualitative interview study of experiences of weight loss maintenance, and the modeling of a justifiable cost for a weight loss maintenance program. Phase 2 included the iterative development of guiding principles, a logic model, and the intervention design and content. Target user and stakeholder panels were established to inform each phase of development, and user testing of successive iterations of the prototype intervention was conducted., Results: This process resulted in a guided self-help ACT-based intervention called SWiM (Supporting Weight Management). SWiM is a 4-month program consisting of weekly web-based sessions for 13 consecutive weeks followed by a 4-week break for participants to reflect and practice their new skills and a final session at week 18. Each session consists of psychoeducational content, reflective exercises, and behavioral experiments. SWiM includes specific sessions on key determinants of weight loss maintenance, including developing skills to manage high-risk situations for lapses, creating new helpful habits, breaking old unhelpful habits, and learning to manage interpersonal relationships and their impact on weight management. A trained, nonspecialist coach provides guidance for the participants through the program with 4 scheduled 30-minute telephone calls and 3 further optional calls., Conclusions: This comprehensive approach facilitated the development of an intervention that is based on scientific theory and evidence for supporting people with weight loss maintenance and is grounded in the experiences of the target users and the context in which it is intended to be delivered. The intervention will be refined based on the findings of a planned pilot randomized controlled trial., (©Rebecca Richards, Rebecca A Jones, Fiona Whittle, Carly A Hughes, Andrew J Hill, Emma R Lawlor, Jennifer Bostock, Sarah Bates, Penny R Breeze, Alan Brennan, Chloe V Thomas, Marie Stubbings, Jennifer Woolston, Simon J Griffin, Amy L Ahern. Originally published in JMIR Formative Research (https://formative.jmir.org), 07.01.2022.)

Active travel (AT) has gained increasing attention as a way of addressing low levels of physical activity. However, little is known regarding the relationship between income and AT. The aim of this study was to investigate characteristics associated with undertaking AT in an adult population and by low- and high-income groups. Data collected from the Physical Activity and the Rejuvenation of Connswater (PARC) study in 2017 were used. Participants were categorised into socio-economic groups according to their weekly household income, and were categorised as participating in 'no' AT or 'some' AT and 'sufficient' AT. Multivariable logistic regression explored characteristics associated with AT in the full cohort, and the low- and high-income groups separately. Variables associated with AT in the low-income group were body mass index (BMI), physical activity self-efficacy, marital status, long term illness, difficulty walking and housing tenure. For the high-income group, BMI, marital status, housing tenure and education were associated with AT. For both income groups, there were consistent positive associations with the action/maintenance phase of the stage of change model across all AT categories. The findings suggest that population sub-groups may benefit from targeted initiatives to support engagement in AT and prevent further widening of inequalities.

Ewing sarcomas are driven by EWS-ETS fusions, most commonly EWS-FLI1, which promotes widespread metabolic reprogramming, including activation of serine biosynthesis. We previously reported that serine biosynthesis is also activated in Ewing sarcoma by the scaffolding protein menin through as yet undefined mechanisms. Here, we investigated whether EWS-FLI1 and/or menin orchestrate serine biosynthesis via modulation of ATF4, a stress-response gene that acts as a master transcriptional regulator of serine biosynthesis in other tumors. Our results show that in Ewing sarcoma, ATF4 levels are high and that ATF4 modulates transcription of core serine synthesis pathway (SSP) genes. Inhibition of either EWS-FLI1 or menin leads to loss of ATF4, and this is associated with diminished expression of SSP transcripts and proteins. We identified and validated an EWS-FLI1 binding site at the ATF4 promoter, indicating that the fusion can directly activate ATF4 transcription. In contrast, our results suggest that menin-dependent regulation of ATF4 is mediated by transcriptional and post-transcriptional mechanisms. Importantly, our data also reveal that the downregulation of SSP genes that occurs in the context of EWS-FLI1 or menin loss is indicative of broader inhibition of ATF4-dependent transcription. Moreover, we find that menin inhibition similarly leads to loss of ATF4 and the ATF4-dependent transcriptional signature in MLL-rearranged B-cell acute lymphoblastic leukemia, extending our findings to another cancer in which menin serves an oncogenic role. IMPLICATIONS: These studies provide new insights into metabolic reprogramming in Ewing sarcoma and also uncover a previously undescribed role for menin in the regulation of ATF4., (©2021 American Association for Cancer Research.)

There is good evidence that behavioural weight management interventions improve physical health; however, the impact on mental health remains unclear. We evaluated the impact of behavioural weight management interventions on mental health-related outcomes in adults with overweight or obesity at intervention-end and 12 months from baseline. Eligible studies were randomized controlled trials (RCTs) or cluster RCTs of adult behavioural weight loss interventions reporting affect, anxiety, binge eating, body image, depression, emotional eating, quality of life, self-esteem and stress. We searched seven databases from inception to 7 May 2019 and included 43 articles reporting 42 RCTs. Eighteen studies were deemed to be at high risk of bias. We conducted random-effects meta-analyses, stratified analyses and meta-regression using Stata. Interventions generated greater improvements than comparators for depression, mental health-related quality of life and self-efficacy at intervention-end and 12 months from baseline. There was no difference between groups for anxiety, overall quality of life, self-esteem or stress at intervention-end. There was insufficient evidence to assess the impact on anxiety, binge eating, body image, emotional eating, affect, life satisfaction, self-esteem or stress at intervention-end and/or 12 months from baseline. Although evidence suggests that interventions benefit some aspects of mental health, high-quality, transparently reported RCTs measuring a range of mental health outcomes over longer durations are required to strengthen the evidence base., (© 2020 The Authors. Obesity Reviews published by John Wiley & Sons Ltd on behalf of World Obesity Federation.)

Background: There is a gradient relationship between socio-economic status and health. We investigated the views and perceptions of health promotion service providers regarding factors that affect lack of engagement in public health initiatives by residents in socio-economically disadvantaged (SED) communities., Methods: We conducted semi-structured interviews with a purposive sample of key providers (n = 15) of community-based health promotion services to elicit their views about engagement-related factors and their experiences of the provision, delivery and impact of health promotion in SED areas. Interviews were analysed using thematic analysis., Results: Failure to (i) recognise within SED communities, socio-cultural norms of health-related behaviour and (ii) communicate to local residents an understanding of complex lifestyle influences appeared to affect adversely service engagement and contribute to the development of negative attitudes towards health promotion. Engagement is more likely when services are delivered within familiar settings, peer support is available, initiatives are organized within existing groups, external incentives are offered and there are options regarding times and locations. Collaborative working between providers and communities facilitates efficient, context-sensitive service delivery., Conclusions: Knowledge of a local community and its socio-environmental context alongside a collaborative, facilitative and tailored approach to delivery are required to ensure successful engagement of SED communities in health promotion., (© The Author(s) 2019. Published by Oxford University Press on behalf of Faculty of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

School-based multi-component physical activity (PA) promotion is advocated; however, research has indicated that a multi-component approach may not always be effective at increasing adolescent PA. Evaluation of the GoActive 12-week multi-component school-based intervention showed no effect on adolescent PA. A mixed-methods process evaluation was embedded to facilitate greater understanding of the results, to elicit subgroup perceptions, and to provide insight into contextual factors influencing intervention implementation. This paper presents the reach, recruitment, dose, and fidelity of GoActive, and identifies challenges to implementation. The process evaluation employed questionnaires (1543 Year 9s), individual interviews (16 Year 9s; 7 facilitators; 9 contact teachers), focus groups (48 Year 9s; 58 mentors), alongside GoActive website analytics and researcher observations. GoActive sessions reached 39.4% of Year 9s. Intervention satisfaction was relatively high for mentors (87.3%) and facilitators (85.7%), but lower for Year 9s (59.5%) and teachers (50%). Intervention fidelity was mixed within and between schools. Mentorship was the most implemented component. Factors potentially contributing to low implementation included ambiguity of the roles subgroups played within intervention delivery, Year 9 engagement, institutional support, and further school-level constraints. Multiple challenges and varying contextual considerations hindered the implementation of GoActive in multiple school sites. Methods to overcome contextual challenges to implementation warrant in-depth consideration and innovative approaches.

While many behavioural weight management programmes are effective in the short-term, post-programme weight regain is common. Overcoming "lapses" and preventing "relapse" has been highlighted as important in weight-loss maintenance, but little is known on how this is achieved. This study aimed to compare the cognitive and behavioural strategies employed to overcome "lapses" and prevent "relapse" by people who had regained weight or maintained weight-loss after participating in a weight management programme. By investigating differences between groups, we intended to identify strategies associated with better weight-loss maintenance. Semi-structured interviews were conducted with 26 participants (58% female) recruited from the 5-year follow-up of the Weight Loss Referrals for Adults in Primary Care (WRAP) trial (evaluation of a commercial weight-loss programme). Participants who had lost ≥5% baseline weight during the active intervention were purposively sampled according to 5-year weight trajectories (n = 16 'Regainers', n = 10 'Maintainers'). Interviews were audio-recorded, transcribed verbatim, and analysed thematically. Key differences in strategies were that Maintainers continued to pay attention to their dietary intake, anticipated and planned for potential lapses in high-risk situations, and managed impulses using distraction techniques. Regainers did not report making plans, used relaxed dietary monitoring, found distraction techniques to be ineffective and appeared to have difficulty navigating food within interpersonal relationships. This study is one of the longest qualitative follow-ups of a weight loss trial to date, offering unique insights into long-term maintenance. Future programmes should emphasize strategies focusing on self-monitoring, planning and managing interpersonal relationships to help participants successfully maintain weight-loss in the longer-term., (© 2020 The Authors. Clinical Obesity published by John Wiley & Sons Ltd on behalf of World Obesity Federation.)

Wnt/β-catenin signaling is active in small subpopulations of Ewing sarcoma cells, and these cells display a more metastatic phenotype, in part due to antagonism of EWS-FLI1-dependent transcriptional activity. Importantly, these β-catenin-activated Ewing sarcoma cells also alter secretion of extracellular matrix (ECM) proteins. We thus hypothesized that, in addition to cell-autonomous mechanisms, Wnt/β-catenin-active tumor cells might contribute to disease progression by altering the tumor microenvironment (TME). Analysis of transcriptomic data from primary patient biopsies and from β-catenin-active versus -nonactive tumor cells identified angiogenic switch genes as being highly and reproducibly upregulated in the context of β-catenin activation. In addition, in silico and in vitro analyses, along with chorioallantoic membrane assays, demonstrated that β-catenin-activated Ewing cells secreted factors that promote angiogenesis. In particular, activation of canonical Wnt signaling leads Ewing sarcoma cells to upregulate expression and secretion of proangiogenic ECM proteins, collectively termed the angiomatrix. Significantly, our data show that induction of the angiomatrix by Wnt-responsive tumor cells is indirect and is mediated by TGF-β. Mechanistically, Wnt/β-catenin signaling antagonizes EWS-FLI1-dependent repression of TGF-β receptor type 2, thereby sensitizing tumor cells to TGF-β ligands. Together, these findings suggest that Wnt/β-catenin-active tumor cells can contribute to Ewing sarcoma progression by promoting angiogenesis in the local TME.

This systematic review and network meta-analysis synthesized evidence on the effects of third-wave cognitive behaviour therapies (3wCBT) on body weight, and psychological and physical health outcomes in adults with overweight or obesity. Studies that included a 3wCBT for the purposes of weight management and measured weight or body mass index (BMI) pre-intervention and ≥ 3 months post-baseline were identified through database searches (MEDLINE, CINAHL, Embase, Cochrane database [CENTRAL], PsycINFO, AMED, ASSIA, and Web of Science). Thirty-seven studies were eligible; 21 were randomized controlled trials (RCT) and included in the network meta-analyses. Risk of bias was assessed using RoB2, and evidence quality was assessed using GRADE. Random-effects pairwise meta-analysis found moderate- to high-quality evidence suggesting that 3wCBT had greater weight loss than standard behavioural treatment (SBT) at post-intervention (standardized mean difference [SMD]: -0.09, 95% confidence interval [CI]: -0.22, 0.04; N = 19; I 2 = 32%), 12 months (SMD: -0.17, 95% CI: -0.36, 0.02; N = 5; I 2 = 33%), and 24 months (SMD: -0.21, 95% CI: -0.42, 0.00; N = 2; I 2 = 0%). Network meta-analysis compared the relative effectiveness of different types of 3wCBT that were not tested in head-to-head trials up to 18 months. Acceptance and commitment therapy (ACT)-based interventions had the most consistent evidence of effectiveness. Only ACT had RCT evidence of effectiveness beyond 18 months. Meta-regression did not identify any specific intervention characteristics (dose, duration, delivery) that were associated with greater weight loss. Evidence supports the use of 3wCBT for weight management, specifically ACT. Larger trials with long-term follow-up are needed to identify who these interventions work for, their most effective components, and the most cost-effective method of delivery., (© 2020 The Authors. Obesity Reviews published by John Wiley & Sons Ltd on behalf of World Obesity Federation.)

Introduction: People with type 2 diabetes (T2D) can improve glycaemic control or even achieve remission through weight loss and reduce their use of medication and risk of cardiovascular disease. The Glucose Lowering through Weight management (GLoW) trial will evaluate whether a tailored diabetes education and behavioural weight management programme (DEW) is more effective and cost-effective than a diabetes education (DE) programme in helping people with overweight or obesity and a recent diagnosis of T2D to lower their blood glucose, lose weight and improve other markers of cardiovascular risk., Methods and Analysis: This study is a pragmatic, randomised, single-blind, parallel group, two-arm, superiority trial. We will recruit 576 adults with body mass index>25 kg/m 2 and diagnosis of T2D in the past 3 years and randomise them to a tailored DEW or a DE programme. Participants will attend measurement appointments at a local general practitioner practice or research centre at baseline, 6 and 12 months. The primary outcome is 12-month change in glycated haemoglobin. The effect of the intervention on the primary outcome will be estimated and tested using a linear regression model (analysis of covariance) including randomisation group and adjusted for baseline value of the outcome and the randomisation stratifiers. Participants will be included in the group to which they were randomised, under the intention-to-treat principle. Secondary outcomes include 6-month and 12-month changes in body weight, body fat percentage, systolic and diastolic blood pressure and lipid profile; probability of achieving good glycaemic control; probability of achieving remission from diabetes; probability of losing 5% and 10% body weight and modelled cardiovascular risk (UKPDS). An intention-to-treat within-trial cost-effectiveness analysis will be conducted from NHS and societal perspectives using participant-level data. Qualitative interviews will be conducted with participants to understand why and how the programme achieved its results and how participants manage their weight after the programme ends., Ethics and Dissemination: Ethical approval was received from East of Scotland Research Ethics Service on 15 May 2018 (18/ES/0048). This protocol (V.3) was approved on 19 June 2019. Findings will be published in peer-reviewed scientific journals and communicated to other stakeholders as appropriate., Trial Registration Number: ISRCTN18399564., Competing Interests: Competing interests: ALA is principal investigator on another publically funded trial where WW provided the intervention at no cost but has received no personal fees. SJG reports grants from Medical Research Council, personal fees from Eli Lilly and personal fees from Janssen, outside this programme of research. AH reports personal fees from Slimming World and the College of Contemporary Health, outside this programme of research. CH reports informal unpaid advice to Thriva. Other Investigators report no conflicts of interest., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

Introduction: The effects of interventions targeting weight loss on physical health are well described, yet the evidence for mental health is less clear. It is essential to better understand the impact of weight management interventions on mental health to optimise care and minimise risk of harm. We will assess the effect of behavioural weight management interventions on mental health in adults with overweight and obesity., Methods and Analysis: The systematic review will follow the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidance. We will include behavioural weight management interventions with a diet and/or physical activity component focusing on weight loss for adults with a body mass index ≥25 kg/m 2 . Randomised controlled trials (RCTs) and cluster RCTs will be the only eligible study designs. Outcomes of interest will be related to mental health. The following databases were searched from inception to 07 May 2019: MEDLINE, Embase, Cochrane database (CENTRAL), PsycINFO, ASSIA, AMED and CINAHL. The search strategy was based on four concepts: (1) adults, defined as ≥18 years, with overweight/obesity, defined as BMI ≥25kg/m², (2) weight management interventions, (3) mental health outcomes and (4) study design. The search was restricted to English-language published papers, with no other restrictions applied. Two stage screening for eligibility will be completed by two independent reviewers, with two independent reviewers completing data extraction and risk of bias assessment. Data permitting, a random-effects meta-analysis of outcomes, subgroup analyses and meta-regression will be conducted. If not appropriate, narrative synthesis and 'levels of evidence' assessment will be completed., Ethics and Dissemination: Ethical approval is not required as primary data will not be collected. The completed systematic review will be disseminated in a peer-reviewed journal, at conferences and contribute towards the lead author's PhD thesis., Prospero Registration Number: CRD42019131659., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

Purpose: To examine adolescent experiences and perspectives of the GoActive intervention (ISRCTN31583496) using mixed methods process evaluation to determine satisfaction with intervention components and interpret adolescents' experiences of the intervention process in order to provide insights for future intervention design., Methods: Participants ( n  = 1542; 13.2 ±  0.4 years, mean ± SD) provided questionnaire data at baseline (shyness, activity level) and post-intervention (intervention acceptability, satisfaction with components). Between-group differences (boys vs. girls and shy/inactive vs. others) were tested with linear regression models, accounting for school clustering. Data from 16 individual interviews (shy/inactive) and 11 focus groups with 48 participants (mean = 4; range 2-7) were thematically coded. Qualitative and quantitative data were merged in an integrative mixed methods convergence matrix, which denoted convergence and dissonance across datasets., Results: Effect sizes for quantitative results were small and may not represent substantial between-group differences. Boys ( vs. girls) preferred class-based sessions ( β  = 0.2, 95% confidence interval (CI): 0.1-0.3); qualitative data suggested that this was because boys preferred competition, which was supported quantitatively ( β  = 0.2, 95%CI: 0.1-0.3). Shy/inactive students did not enjoy the competition ( β  = -0.3, 95%CI: -0.5 to -0.1). Boys enjoyed trying new activities more ( β  = 0.1, 95%CI: 0.1-0.2); qualitative data indicated a desire to try new activities across all subgroups but identified barriers to choosing unfamiliar activities with self-imposed choice restriction leading to boredom. Qualitative data highlighted critique of mentorship; adolescents liked the idea, but older mentors did not meet expectations., Conclusion: We interpreted adolescent perspectives of intervention components and implementation to provide insights into future complex interventions aimed at increasing young people's physical activity in school-based settings. The intervention component mentorship was liked in principle, but implementation issues undesirably impacted satisfaction; competition was disliked by girls and shy/inactive students. The results highlight the importance of considering gender differences in preference of competition and extensive mentorship training., (© 2019 Published by Elsevier B.V. on behalf of Shanghai University of Sport.)

Ewing sarcoma is a bone tumor most commonly diagnosed in adolescents and young adults. Survival for patients with recurrent or metastatic Ewing sarcoma is dismal and there is a dire need to better understand the mechanisms of cell metastasis specific to this disease. Our recent work demonstrated that microenvironmental stress leads to increased Ewing sarcoma cell invasion through Src activation. Additionally, we have shown that the matricellular protein tenascin C (TNC) promotes metastasis in Ewing sarcoma. A major role of both TNC and Src is mediation of cell-cell and cell-matrix interactions resulting in changes in cell motility, invasion, and adhesion. However, it remains largely unknown, if and how, TNC and Src are linked in these processes. We hypothesized that TNC is a positive regulator of invadopodia formation in Ewing sarcoma through its ability to activate Src. We demonstrate here that both tumor cell endogenous and exogenous TNC can enhance Src activation and invadopodia formation in Ewing sarcoma. We found that microenvironmental stress upregulates TNC expression and this is dampened with application of the Src inhibitor dasatinib, suggesting that TNC expression and Src activation cooperate to promote the invasive phenotype. This work reports the impact of stress-induced TNC expression on enhancing cell invadopodia formation, provides evidence for a feed forward loop between TNC and Src to promote cell metastatic behavior, and highlights a pathway by which microenvironment-driven TNC expression could be therapeutically targeted in Ewing sarcoma., (Copyright © 2019 Neoplasia Press, Inc. Published by Elsevier Inc. All rights reserved.)

Background: Insufficient physical activity (PA) is a major public health issue. Whilst PA is an important contributor to disease prevention, engagement in PA decreases with age, particularly among women in socio-economically disadvantaged areas. Research using existing support networks to engage 'hard to reach' populations in PA interventions is sparse. We developed and tested the feasibility of a PA-promoting intervention for older women within existing community groups in socio-economically disadvantaged areas., Methods: The Medical Research Council guidelines for complex interventions were used to guide the intervention's development. We recruited participants (n = 40) from older (aged ≥50 years) women's groups from four different community centres. A 12-week programme was delivered during existing sessions, informed by Social Practice Theory. The sessions provided education about PA, social support in the form of a PA 'buddy', group discussion and follow-up telephone calls, as well as printed information about local opportunities to participate in PA. The main uncertainties tested were rates of participant recruitment, retention, and completion of assessments of PA by accelerometry and of mental health using the Hospital Anxiety and Depression Scale (HADS). Intervention acceptability was assessed by questionnaire, and focus group interviews elicited participants' views about the intervention. Qualitative data were subjected to framework analysis., Results: The recruitment rate was high; 87% (n = 40/46) of women consented to participate, and 78% (n = 31) attended all education sessions. Uptake of follow-up telephone calls and PA 'buddies' was low. Few participants provided valid accelerometer data, but 63% (n=25) completed the HADS questionnaire at all time points. The printed materials and education sessions were viewed positively; telephone calls and 'buddy' support were not valued. Participants believed that organised group activities would lead to increased PA engagement, and whilst participants disliked wearing a waist accelerometer, they thought that regular PA feedback would facilitate necessary goal-setting., Conclusions: High recruitment and retention rates suggest that use of existing social support groups is an acceptable and attractive method of delivering a PA intervention to this population. A randomised controlled trial of the intervention appears feasible, but its design requires refinement of the social support component, facilitation of goal-setting and reconsideration of the assessment of PA., Trial Registration: ClinicalTrials.gov, NCT02880449 . Registered on 26 August 2016.

Ewing sarcomas predominantly arise in pelvic and stylopod bones (i.e., femur and humerus), likely as a consequence of EWS-FLI1 oncogene-induced transformation of mesenchymal stem/progenitor cells (MSCs). MSCs located in the embryonic superficial zone cells (eSZ) of limbs express anatomically distinct posterior Hox genes. Significantly, high expression of posterior HOXD genes, especially HOXD13 , is a hallmark of Ewing sarcoma. These data drove our hypothesis that Hox genes in posterior skeleton MSCs contribute to Ewing sarcoma tumorigenesis. We isolated eSZ cells from stylopod and zeugopod (i.e., tibia/fibula, radius/ulna) bones, from wild-type and Hoxd13 mutant embryos, and tested the impact of EWS-FLI1 transduction on cell proliferation, gene expression, and tumorigenicity. Our data demonstrate that both stylopod and zeugopod eSZ cells tolerate EWS-FLI1 but that stylopod eSZ cells are relatively more susceptible, demonstrating changes in proliferation and gene expression consistent with initiation of malignant transformation. Significantly, loss of Hoxd13 had no impact, showing that it is dispensable for the initiation of EWS-FLI1 -induced transformation in mouse MSCs. These findings show that MSCs from anatomically distinct sites are differentially susceptible to EWS-FLI1-induced transformation, supporting the premise that the dominant presentation of Ewing sarcoma in pelvic and stylopod bones is attributable to anatomically-defined differences in MSCs.

Background: Previous studies have shown that aberrant activation of the Wnt/β-catenin pathway is associated with many malignant neoplasms. This includes some soft-tissue sarcoma phenotypes, most notably synovial sarcoma, implicating potential targets for novel molecular therapies., Objective: We investigate the level of Wnt/β-catenin pathway activation present in leiomyosarcomas relative to synovial sarcomas, using expression of LEF1 and β-catenin as surrogates., Methods: Cancer outlier profile analysis was performed on messenger RNA expression datasets in Oncomine (70 synovial sarcomas, 178 leiomyosarcomas). Results for LEF1 and β-catenin messenger RNA expression were reported in terms of median-centered intensity. Separate immunohistochemical studies were performed on tissue microarrays created from 77 synovial sarcomas and 89 leiomyosarcomas using antibodies to LEF1 and β-catenin. Tumors with unequivocal strong nuclear staining involving ⩾5% of cells were interpreted as positive., Results: Cancer outlier profile analysis demonstrated a higher level of LEF1 messenger RNA expression in synovial sarcomas than in leiomyosarcomas ( p  < 0.0001), but showed no significant difference in β-catenin messenger RNA expression ( p  = 0.868). Immunohistochemistry showed most synovial sarcomas had strong nuclear expression of LEF1 (79%) and β-catenin (84%), while a small minority of leiomyosarcomas had strong nuclear expression of LEF1 (5%) and β-catenin (6%)., Conclusion: These results provide further evidence that aberrant activation of the Wnt/β-catenin pathway is present in most synovial sarcomas, but not in most leiomyosarcomas. While targeting the constituents of this pathway might be effective in the treatment of synovial sarcomas, it is not likely to be an effective strategy in the treatment of leiomyosarcomas., Competing Interests: Conflict of interest: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Cardiovascular disease (CVD) is the leading cause of death worldwide, and its prevalence is increasing; with limited healthcare resources, secondary prevention programmes outside traditional hospital settings are needed, but their effectiveness is unclear. We aimed to assess the effectiveness of secondary prevention cardiovascular risk reduction programmes delivered in venues situated within the community on modification of behavioural risk factors. We searched five databases (MEDLINE, EMBASE, CINAHL, PsycINFO, Cochrane library) to identify trials of health behaviour interventions for adults with CVD in community-based venues. Primary outcomes were changes in physical activity, diet, smoking and/or alcohol consumption. Two reviewers independently assessed articles for eligibility and risk of bias; statistical analysis used Revman v5.3. Of 5905 articles identified, 41 articles (38 studies) (n = 7970) were included. Interventions were mainly multifactorial, educational, psychological and physical activity-based. Meta-analyses identified increased steps/week (Mean Difference (MD): 7480; 95% CI 1,940, 13,020) and minutes of physical activity/week (MD: 59.96; 95% CI 15.67, 104.25) associated with interventions. There was some evidence for beneficial effects on peak VO 2 , blood pressure, total cholesterol and mental health. Variation in outcome measurements reported for other behavioural risk factors limited our ability to perform meta-analyses. Effective interventions were based in homes, general practices or outpatient settings, individually tailored and often multicomponent with a theoretical framework. Our review identified evidence that interventions for secondary CVD prevention, delivered in various community-based venues, have positive effects on physical activity; such opportunities should be promoted by health professionals., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Multi-agent chemotherapeutic regimes remain the cornerstone treatment for Ewing sarcoma, the second most common bone malignancy diagnosed in pediatric and young adolescent populations. We have reached a therapeutic ceiling with conventional cytotoxic agents, highlighting the need to adopt novel approaches that specifically target the drivers of Ewing sarcoma oncogenesis. As KDM1A/ l ysine- s pecific d emethylase 1 (LSD1) is highly expressed in Ewing sarcoma cell lines and tumors, with elevated expression levels associated with worse overall survival ( P = 0.033), this study has examined biomarkers of sensitivity and mechanisms of cytotoxicity to targeted KDM1A inhibition using SP-2509 (reversible KDM1A inhibitor). We report, that innate resistance to SP-2509 was not observed in our Ewing sarcoma cell line cohort ( n = 17; IC 50 range, 81 -1,593 nmol/L), in contrast resistance to the next-generation KDM1A irreversible inhibitor GSK-LSD1 was observed across multiple cell lines (IC 50 > 300 μmol/L). Although TP53/STAG2/CDKN2A status and basal KDM1A mRNA and protein levels did not correlate with SP-2509 response, induction of KDM1B following SP-2509 treatment was strongly associated with SP-2509 hypersensitivity. We show that the transcriptional profile driven by SP-2509 strongly mirrors KDM1A genetic depletion. Mechanistically, RNA-seq analysis revealed that SP-2509 imparts robust apoptosis through engagement of the endoplasmic reticulum stress pathway. In addition, ETS1/HIST1H2BM were specifically induced/repressed, respectively following SP-2509 treatment only in our hypersensitive cell lines. Together, our findings provide key insights into the mechanisms of SP-2509 cytotoxicity as well as biomarkers that can be used to predict KDM1A inhibitor sensitivity in Ewing sarcoma. Mol Cancer Ther; 17(9); 1902-16. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Introduction: Behavioural and cognitive behavioural programmes are commonly used to assist with weight management, but there is considerable scope to improve their effectiveness, particularly in the longer term. Third-wave cognitive behaviour therapies (CBTs) have this potential and are increasingly used. This systematic review will assess the effect of third-wave CBTs for weight management on weight, psychological and physical health outcomes in adults with overweight or obesity., Methods and Analysis: The systematic review will be reported according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidance. We will include studies of any third-wave CBTs focusing on weight loss or weight maintenance for adults with a body mass index (BMI) ≥25kg/m 2 . Eligible study designs will be randomised control trials, non-randomised trials, prospective cohort and case series. Outcomes of interest will be body weight/BMI, psychological and physical health, and adherence. We will search the following databases from inception to 16 January 2018: MEDLINE, CINAHL, Embase, Cochrane database (CENTRAL), PsycINFO, AMED, ASSIA and Web of Science. The search strategy will be based on the concepts: (1) third-wave CBTs and (2) overweight, obesity or weight management. No restrictions will be applied. We will search reference lists of relevant reviews and included articles. Two independent reviewers will screen articles for eligibility using a two-stage process. Two independent reviewers will extract data, assess risk of bias using Risk of Bias 2.0, Risk of Bias in Non-randomised studies of Interventions or Risk of Bias in Non-randomised Studies of Exposures checklist and assess quality using the Grading of Recommendations Assessment, Development and Evaluation tool. A random-effects network meta-analysis of outcomes, and sub-group analyses and meta-regression will be conducted, where data permit. If not appropriate, a narrative synthesis will be undertaken., Ethics and Dissemination: Ethical approval is not required as no primary data will be collected. The completed systematic review will be disseminated in a peer-reviewed journal, presented at conferences and used to inform the development of a weight management programme., Prospero Registration Number: CRD42018088255., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2018. Re-use permitted under CC BY. Published by BMJ.)

Developmental transcription programs are epigenetically regulated by multi-protein complexes, including the menin- and MLL-containing trithorax (TrxG) complexes, which promote gene transcription by depositing the H3K4me3 activating mark at target gene promoters. We recently reported that in Ewing sarcoma, MLL1 (lysine methyltransferase 2A, KMT2A) and menin are overexpressed and function as oncogenes. Small molecule inhibition of the menin-MLL interaction leads to loss of menin and MLL1 protein expression, and to inhibition of growth and tumorigenicity. Here, we have investigated the mechanistic basis of menin-MLL-mediated oncogenic activity in Ewing sarcoma. Bromouridine sequencing (Bru-seq) was performed to identify changes in nascent gene transcription in Ewing sarcoma cells, following exposure to the menin-MLL interaction inhibitor MI-503. Menin-MLL inhibition resulted in early and widespread reprogramming of metabolic processes. In particular, the serine biosynthetic pathway (SSP) was the pathway most significantly affected by MI-503 treatment. Baseline expression of SSP genes and proteins (PHGDH, PSAT1, and PSPH), and metabolic flux through the SSP were confirmed to be high in Ewing sarcoma. In addition, inhibition of PHGDH resulted in reduced cell proliferation, viability, and tumor growth in vivo, revealing a key dependency of Ewing sarcoma on the SSP. Loss of function studies validated a mechanistic link between menin and the SSP. Specifically, inhibition of menin resulted in diminished expression of SSP genes, reduced H3K4me3 enrichment at the PHGDH promoter, and complete abrogation of de novo serine and glycine biosynthesis, as demonstrated by metabolic tracing studies with 13 C-labeled glucose. These data demonstrate that the SSP is highly active in Ewing sarcoma and that its oncogenic activation is maintained, at least in part, by menin-dependent epigenetic mechanisms involving trithorax complexes. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

In this Letter, the sentence beginning "This work was funded…." in the Acknowledgements should have read "CPRIT (RP140105) to J.C.R." rather than "CPRIT (RP150445) to J.C.R." This error has been corrected online.

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard subcutaneous flank models include procedural ease and the ability to monitor tumor progression and response without invasive imaging. Such models are often inconsistent in translational clinical trials and have limited biologically relevant characteristics with low proclivity to produce metastasis, as there is a lack of a native microenvironment. In comparison, orthotopic xenograft models at native tumor sites have been shown to mimic the tumor microenvironment and replicate important disease characteristics such as distant metastatic spread. These models often require tedious surgical procedures with prolonged anesthetic time and recovery periods. To address this, cancer researchers have recently utilized ultrasound-guided injection techniques to establish cancer xenograft models for preclinical experiments, which allows for rapid and reliable establishment of tissue-directed murine models. Ultrasound visualization also provides a noninvasive method for longitudinal assessment of tumor engraftment and growth. Here, we describe the method for ultrasound-guided injection of cancer cells, utilizing the adrenal gland for NB and renal sub capsule for ES. This minimally invasive approach overcomes tedious open surgery implantation of cancer cells in tissue-specific locations for growth and metastasis, and abates morbid recovery periods. We describe the utilization of both established cell lines and patient derived cell lines for orthotopic injection. Pre-made commercial kits are available for tumor dissociation and luciferase tagging of cells. Injection of cell suspension using image-guidance provides a minimally invasive and reproducible platform for the creation of preclinical models. This method is utilized to create reliable preclinical models for other cancers such as bladder, liver and pancreas exemplifying its untapped potential for numerous cancer models.

Tumor: tumor microenvironment (TME) interactions are critical for tumor progression and the composition and structure of the local extracellular matrix (ECM) are key determinants of tumor metastasis. We recently reported that activation of Wnt/beta-catenin signaling in Ewing sarcoma cells induces widespread transcriptional changes that are associated with acquisition of a metastatic tumor phenotype. Significantly, ECM protein-encoding genes were found to be enriched among Wnt/beta-catenin induced transcripts, leading us to hypothesize that activation of canonical Wnt signaling might induce changes in the Ewing sarcoma secretome. To address this hypothesis, conditioned media from Ewing sarcoma cell lines cultured in the presence or absence of Wnt3a was collected for proteomic analysis. Label-free mass spectrometry was used to identify and quantify differentially secreted proteins. We then used in silico databases to identify only proteins annotated as secreted. Comparison of the secretomes of two Ewing sarcoma cell lines revealed numerous shared proteins, as well as a degree of heterogeneity, in both basal and Wnt-stimulated conditions. Gene set enrichment analysis of secreted proteins revealed that Wnt stimulation reproducibly resulted in increased secretion of proteins involved in ECM organization, ECM receptor interactions, and collagen formation. In particular, Wnt-stimulated Ewing sarcoma cells up-regulated secretion of structural collagens, as well as matricellular proteins, such as the metastasis-associated protein, tenascin C (TNC). Interrogation of published databases confirmed reproducible correlations between Wnt/beta-catenin activation and TNC and COL1A1 expression in patient tumors. In summary, this first study of the Ewing sarcoma secretome reveals that Wnt/beta-catenin activated tumor cells upregulate secretion of ECM proteins. Such Wnt/beta-catenin mediated changes are likely to impact on tumor: TME interactions that contribute to metastatic progression., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Ewing sarcoma is an aggressive paediatric cancer of the bone and soft tissue. It results from a chromosomal translocation, predominantly t(11;22)(q24:q12), that fuses the N-terminal transactivation domain of the constitutively expressed EWSR1 protein with the C-terminal DNA binding domain of the rarely expressed FLI1 protein. Ewing sarcoma is highly sensitive to genotoxic agents such as etoposide, but the underlying molecular basis of this sensitivity is unclear. Here we show that Ewing sarcoma cells display alterations in regulation of damage-induced transcription, accumulation of R-loops and increased replication stress. In addition, homologous recombination is impaired in Ewing sarcoma owing to an enriched interaction between BRCA1 and the elongating transcription machinery. Finally, we uncover a role for EWSR1 in the transcriptional response to damage, suppressing R-loops and promoting homologous recombination. Our findings improve the current understanding of EWSR1 function, elucidate the mechanistic basis of the sensitivity of Ewing sarcoma to chemotherapy (including PARP1 inhibitors) and highlight a class of BRCA-deficient-like tumours.

Neuroblastoma is a cancer of neural crest stem cell (NCSC) lineage. Signaling pathways that regulate NCSC differentiation have been implicated in neuroblastoma tumorigenesis. This is exemplified by MYCN oncogene targets that balance proliferation, differentiation, and cell death similarly in normal NCSC and in high-risk neuroblastoma. Our previous work discovered a survival mechanism by which MYCN-amplified neuroblastoma circumvents cell death by upregulating components of the error-prone non-canonical alternative nonhomologous end-joining (alt-NHEJ) DNA repair pathway. Similar to proliferating stem cells, high-risk neuroblastoma cells have enhanced DNA repair capacity, overcoming DNA damage with higher repair efficiency than somatic cells. Adequate DNA maintenance is required for lineage protection as stem cells proliferate and during tumor progression to overcome oncogene-induced replication stress. On this basis, we hypothesized that alt-NHEJ overexpression in neuroblastoma is a cancer cell survival mechanism that originates from DNA repair systems of NCSC, the presumed progenitor cell of origin. A human NCSC model was generated in which inducible MYCN triggered an immortalized phenotype capable of forming metastatic neuroectodermal tumors in mice, resembling human neuroblastoma. Critical alt-NHEJ components (DNA Ligase III, DNA Ligase I, and Poly [ADP-ribose polymerase 1]) were highly expressed in normal early NCSC, and decreased as cells became terminally differentiated. Constitutive MYCN expression maintained high alt-NHEJ protein expression, preserving the expression pattern of the immature neural phenotype. siRNA knockdown of alt-NHEJ components reversed MYCN effects on NCSC proliferation, invasion, and migration. DNA Ligase III, Ligase I, and PARP1 silencing significantly decreased neuroblastoma markers expression (TH, Phox2b, and TRKB). These results utilized the first human NCSC model of neuroblastoma to uncover an important link between MYCN and alt-NHEJ expression in developmental tumor initiation, setting precedence to investigate alt-NHEJ repair mechanics in neuroblastoma DNA maintenance.

Background: Advances in cancer therapeutics depend on reliable in vivo model systems. To develop biologically relevant xenografts, ultrasound was utilized for tissue-directed implantation of neuroblastoma (NB) cell line and patient-derived tumors in the adrenal gland, and for renal subcapsular engraftment of Ewing's sarcoma (ES)., Materials and Methods: NB xenografts were established by direct adrenal injection of luciferase-transfected NB cell lines (IMR32, SH-SY5Y, SK-N-BE2) or NB patient-derived tumor cells (UMNBL001, UMNBL002). ES xenografts were established by renal subcapsular injection of TC32, A673, CHLA-25, or A4573 cells. Progression was monitored by in vivo imaging., Results: Tumors progressed to local disease with metastasis evident by 5 weeks. Metastatic sites included cortical bone, lung, liver, and lymph nodes. Xenografted tumors retained immunochemical features of the original cancer., Conclusion: Human NB adrenal xenografts, including two patient-derived orthotopic, and ES renal subcapsular xenografts were established by ultrasound without open surgery. Tissue-directed implantation is an effective technique for developing metastatic preclinical models., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Developmental transcription programs are epigenetically regulated by the competing actions of polycomb and trithorax (TrxG) protein complexes, which repress and activate genes, respectively. Ewing sarcoma is a developmental tumor that is associated with widespread de-regulation of developmental transcription programs, including HOX programs. Posterior HOXD genes are abnormally over-expressed by Ewing sarcoma and HOXD13, in particular, contributes to the tumorigenic phenotype. In MLL1 fusion-driven leukemia, aberrant activation of HOXA genes is epigenetically mediated by the TrxG complex and HOXA gene expression and leukemogenesis are critically dependent on the protein-protein interaction between the TrxG proteins MLL1 and menin. Based on these data, we investigated whether posterior HOXD gene activation and Ewing sarcoma tumorigenicity are similarly mediated by and dependent on MLL1 and/or menin. Our findings demonstrate that Ewing sarcomas express high levels of both MLL1 and menin and that continued expression of both proteins is required for maintenance of tumorigenicity. In addition, exposure of Ewing sarcoma cells to MI-503, an inhibitor of the MLL1-menin protein-protein interaction developed for MLL1-fusion driven leukemia, leads to loss of tumorigenicity and down-regulated expression of the posterior HOXD gene cluster. Together these data demonstrate an essential role for MLL1 and menin in mediating tumor maintenance and posterior HOXD gene activation in Ewing sarcoma. A critical dependency of these tumors on the MLL1-menin interaction presents a potentially novel therapeutic target.

Melanoma is the most dangerous form of skin cancer with the majority of deaths arising from metastatic disease. Evidence implicates Rho-activated gene transcription in melanoma metastasis mediated by the nuclear localization of the transcriptional coactivator, myocardin-related transcription factor (MRTF). Here, we highlight a role for Rho and MRTF signaling and its reversal by pharmacologic inhibition using in vitro and in vivo models of human melanoma growth and metastasis. Using two cellular models of melanoma, we clearly show that one cell type, SK-Mel-147, is highly metastatic, has high RhoC expression, and MRTF nuclear localization and activity. Conversely, SK-Mel-19 melanoma cells have low RhoC expression, and decreased levels of MRTF-regulated genes. To probe the dependence of melanoma aggressiveness to MRTF transcription, we use a previously developed small-molecule inhibitor, CCG-203971, which at low micromolar concentrations blocks nuclear localization and activity of MRTF-A. In SK-Mel-147 cells, CCG-203971 inhibits cellular migration and invasion, and decreases MRTF target gene expression. In addition, CCG-203971-mediated inhibition of the Rho/MRTF pathway significantly reduces cell growth and clonogenicity and causes G 1 cell-cycle arrest. In an experimental model of melanoma lung metastasis, the RhoC-overexpressing melanoma cells (SK-Mel-147) exhibited pronounced lung colonization compared with the low RhoC-expressing SK-Mel-19. Furthermore, pharmacologic inhibition of the MRTF pathway reduced both the number and size of lung metastasis resulting in a marked reduction of total lung tumor burden. These data link Rho and MRTF-mediated signaling with aggressive phenotypes and support targeting the MRTF transcriptional pathway as a novel approach to melanoma therapeutics. Mol Cancer Ther; 16(1); 193-204. ©2016 AACR., (©2016 American Association for Cancer Research.)

BMI1 is a core component of the polycomb repressive complex 1 (PRC1) and emerging data support a role of BMI1 in cancer. The central domain of BMI1 is involved in protein-protein interactions and is essential for its oncogenic activity. Here, we present the structure of BMI1 bound to the polyhomeotic protein PHC2 illustrating that the central domain of BMI1 adopts an ubiquitin-like (UBL) fold and binds PHC2 in a β-hairpin conformation. Unexpectedly, we find that the UBL domain is involved in homo-oligomerization of BMI1. We demonstrate that both the interaction of BMI1 with polyhomeotic proteins and homo-oligomerization via UBL domain are necessary for H2A ubiquitination activity of PRC1 and for clonogenic potential of U2OS cells. Here, we also emphasize need for joint application of NMR spectroscopy and X-ray crystallography to determine the overall structure of the BMI1-PHC2 complex., Competing Interests: Drs Grembecka and Cierpicki receive research support from Kura Oncology. They are also receiving compensation as members of the scientific advisory board of Kura Oncology, and they have an equity ownership in the company. Other co-authors declare no potential conflict of interest.

Background: The prognostic significance of having extraskeletal (EES) versus skeletal Ewing sarcoma (ES) in the setting of modern chemotherapy protocols is unknown. The purpose of this study was to compare the clinical characteristics, biologic features, and outcomes for patients with EES and skeletal ES., Methods: Patients had localized ES and were treated on two consecutive protocols using five-drug chemotherapy (INT-0154 and AEWS0031). Patients were analyzed based on having an extraskeletal (n = 213) or skeletal (n = 826) site of tumor origin. Event-free survival (EFS) was estimated using the Kaplan-Meier method, compared using the log-rank test, and modeled using Cox multivariate regression., Results: Patients with extraskeletal ES (EES) were more likely to have axial tumors (72% vs. 55%; P < 0.001), less likely to have tumors >8 cm (9% vs. 17%; P < 0.01), and less likely to be white (81% vs. 87%; P < 0.001) compared to patients with skeletal ES. There was no difference in key genomic features (type of EWSR1 translocation, TP53 mutation, CDKN2A mutation/loss) between groups. After controlling for age, race, and primary site, EES was associated with superior EFS (hazard ratio = 0.69; 95% confidence interval: 0.50-0.95; P = 0.02). Among patients with EES, age ≥18, nonwhite race, and elevated baseline erythrocyte sedimentation rate were independently associated with inferior EFS., Conclusion: Clinical characteristics, but not key tumor genomic features, differ between EES and skeletal ES. Extraskeletal origin is a favorable prognostic factor, independent of age, race, and primary site., Competing Interests: Statement None of the authors have any conflicts of interest related to this work or its publication. Author S.L.L. has a leadership role and stock/ownership interest in Salarius Pharmaceuticals, however, again, this is not a conflict for this study., (© 2016 Wiley Periodicals, Inc.)