Cohesin-mediated 3D contacts tune enhancer-promoter regulation.

Enhancers are key drivers of gene regulation thought to act via 3D physical interactions with the promoters of their target genes. However, genome-wide depletions of architectural proteins such as cohesin result in only limited changes in gene expression, despite a loss of contact domains and loops. Consequently, the role of cohesin and 3D contacts in enhancer function remains debated. Here, we developed CRISPRi of regulatory elements upon degron operation (CRUDO), a novel approach to measure how changes in contact frequency impact enhancer effects on target genes by perturbing enhancers with CRISPRi and measuring gene expression in the presence or absence of cohesin. We systematically perturbed all 1,039 candidate enhancers near five cohesin-dependent genes and identified 34 enhancer-gene regulatory interactions. Of 26 regulatory interactions with sufficient statistical power to evaluate cohesin dependence, 18 show cohesin-dependent effects. A decrease in enhancer-promoter contact frequency upon removal of cohesin is frequently accompanied by a decrease in the regulatory effect of the enhancer on gene expression, consistent with a contact-based model for enhancer function. However, changes in contact frequency and regulatory effects on gene expression vary as a function of distance, with distal enhancers (e.g., >50Kb) experiencing much larger changes than proximal ones (e.g., <50Kb). Because most enhancers are located close to their target genes, these observations can explain how only a small subset of genes - those with strong distal enhancers - are sensitive to cohesin. Together, our results illuminate how 3D contacts, influenced by both cohesin and genomic distance, tune enhancer effects on gene expression.

Factors associated with transition to a nursing home in older adults living in naturally occurring retirement communities.

BACKGROUND: Naturally occurring retirement communities (NORCs) are geographic areas (generally high-rise buildings or neighborhoods) that have a high concentration of individuals 65 years and older. Supportive service programs in NORCs can address resident needs and delay nursing home (NH) admission but understanding what factors are associated with NORC residents requiring NH admission is needed to tailor such programs. Our aim was to examine individual- and neighborhood-level factors associated with NH wait-list status in NORC residents in Ontario. METHODS: We conducted a population-based, cross-sectional study of Ontario adults 65 years of age or older living in a NORC building as of January 1, 2020, by linking a provincial registry of NORC high-rise buildings with health administrative data. Older adults were classified as being on the NH wait-list if they had an open application for a NH on the index date. We conducted a multilevel logistic regression analysis using generalized estimating equations to determine individual- and neighborhood-level factors associated with NH wait-list status, including sociodemographic, clinical, healthcare use, and building factors. We explored the role of sex and age through stratification by sex (male, female) and age (65-80 and 80+ years). RESULTS: Among 220,864 NORC residents, 4710 individuals (2.1%) were on the NH wait-list. Female sex, older age, immigrant status, dementia diagnosis, receiving homecare, multimorbidity, and polypharmacy (five or more unique drug names) were associated with an increased odds of wait-list status. Several neighborhood-level variables were associated with a significantly increased likelihood of wait-list status, including low income, high dependency, high ethnic diversity, and living in a building with supports. CONCLUSION: NORC supportive service programs can be tailored to account for the factors associated with NH wait-list status, allowing NORC residents who are living in the community to age in their desired place and achieve optimal health outcomes.

An encyclopedia of enhancer-gene regulatory interactions in the human genome.

Identifying transcriptional enhancers and their target genes is essential for understanding gene regulation and the impact of human genetic variation on disease1-6. Here we create and evaluate a resource of >13 million enhancer-gene regulatory interactions across 352 cell types and tissues, by integrating predictive models, measurements of chromatin state and 3D contacts, and largescale genetic perturbations generated by the ENCODE Consortium7. We first create a systematic benchmarking pipeline to compare predictive models, assembling a dataset of 10,411 elementgene pairs measured in CRISPR perturbation experiments, >30,000 fine-mapped eQTLs, and 569 fine-mapped GWAS variants linked to a likely causal gene. Using this framework, we develop a new predictive model, ENCODE-rE2G, that achieves state-of-the-art performance across multiple prediction tasks, demonstrating a strategy involving iterative perturbations and supervised machine learning to build increasingly accurate predictive models of enhancer regulation. Using the ENCODE-rE2G model, we build an encyclopedia of enhancer-gene regulatory interactions in the human genome, which reveals global properties of enhancer networks, identifies differences in the functions of genes that have more or less complex regulatory landscapes, and improves analyses to link noncoding variants to target genes and cell types for common, complex diseases. By interpreting the model, we find evidence that, beyond enhancer activity and 3D enhancer-promoter contacts, additional features guide enhancerpromoter communication including promoter class and enhancer-enhancer synergy. Altogether, these genome-wide maps of enhancer-gene regulatory interactions, benchmarking software, predictive models, and insights about enhancer function provide a valuable resource for future studies of gene regulation and human genetics.

A synthetic lethal screen for Snail-induced enzalutamide resistance identifies JAK/STAT signaling as a therapeutic vulnerability in prostate cancer.

Despite substantial improvements in the treatment landscape of prostate cancer, the evolution of hormone therapy-resistant and metastatic prostate cancer remains a major cause of cancer-related death globally. The mainstay of treatment for advanced prostate cancer is targeting of androgen receptor signaling, including androgen deprivation therapy plus second-generation androgen receptor blockade (e.g., enzalutamide, apalutamide, darolutamide), and/or androgen synthesis inhibition (abiraterone). While these agents have significantly prolonged the lives of patients with advanced prostate cancer, is nearly universal. This therapy resistance is mediated by diverse mechanisms, including both androgen receptor-dependent mechanisms, such as androgen receptor mutations, amplifications, alternative splicing, and amplification, as well as non-androgen receptor-mediated mechanisms, such as lineage plasticity toward neuroendocrine-like or epithelial-mesenchymal transition (EMT)-like lineages. Our prior work identified the EMT transcriptional regulator Snail as critical to hormonal therapy resistance and is commonly detected in human metastatic prostate cancer. In the current study, we sought to interrogate the actionable landscape of EMT-mediated hormone therapy resistant prostate cancer to identify synthetic lethality and collateral sensitivity approaches to treating this aggressive, therapy-resistant disease state. Using a combination of high-throughput drug screens and multi-parameter phenotyping by confluence imaging, ATP production, and phenotypic plasticity reporters of EMT, we identified candidate synthetic lethalities to Snail-mediated EMT in prostate cancer. These analyses identified multiple actionable targets, such as XPO1, PI3K/mTOR, aurora kinases, c-MET, polo-like kinases, and JAK/STAT as synthetic lethalities in Snail+ prostate cancer. We validated these targets in a subsequent validation screen in an LNCaP-derived model of resistance to sequential androgen deprivation and enzalutamide. This follow-up screen provided validation of inhibitors of JAK/STAT and PI3K/mTOR as therapeutic vulnerabilities for both Snail+ and enzalutamide-resistant prostate cancer.

Primary Care Clinical Volumes, Cholesterol Testing, and Cardiovascular Outcomes.

BACKGROUND: It is unknown whether the annual number of primary care physician (PCP) unique outpatient assessments, which we refer to as clinical volume, translates into better cardiovascular preventive care. We examined the relationship between PCP outpatient clinical volumes and cholesterol testing and major adverse cardiovascular event rates among guideline-recommended eligible patients. METHODS: This was a retrospective cohort study conducted as part of the Cardiovascular Health in Ambulatory Care Research Team (CANHEART) cohort, a population-based cohort of almost all adult residents of Ontario, Canada, followed from 2008 to 2012. For each clinical volume quintile, we compared cholesterol testing and major adverse cardiovascular events, defined as time to first event of cardiovascular death, nonfatal myocardial infarction, or nonfatal stroke. RESULTS: The 10,037 PCPs evaluated had an annualized median volume of 2303 clinical encounters (IQR 1292-3680). Among 4,740,380 patients, 84% underwent guideline-concordant cholesterol testing at least once over 5 years, ranging from 73% with the lowest clinical volume quintile physicians to 86% with the highest. After multivariable adjustment, there was a 10.5% relative increase in the probability of cholesterol testing for every doubling of clinical volumes (95% CI 9.7-11.4; P < 0.001). Patients treated by the lowest volume quintile physicians had the highest rate of major adverse cardiovascular outcomes (compared with the highest volume quintile physicians: adjusted HR 1.15, 95% CI 1.10-1.21; P < 0.001). CONCLUSIONS: Patients of physicians with the lowest clinical volumes received less frequent cholesterol testing and had the highest rate of incident cardiovascular events. Further research investigating the drivers of this relationship is warranted.

Eligibility and Implementation of Rivaroxaban for Secondary Prevention of Atherothrombosis in Clinical Practice-Insights From the CANHEART Study.

Background The COMPASS (Cardiovascular Outcomes for People Using Anticoagulation Strategies) trial decreased major adverse cardiovascular events with very low-dose rivaroxaban and aspirin in patients with coronary artery disease and peripheral artery disease. We examined the eligibility and potential real-world impact of this strategy on the COMPASS-eligible population. Methods and Results COMPASS eligibility criteria were applied to the CANHEART (Cardiovascular Health in Ambulatory Care Research Team) registry, a population-based cohort of Ontario adults. We compared 5-year major adverse cardiovascular events and major bleeding rates stratified by COMPASS eligibility and by clinical risk factors. We applied COMPASS trial rivaroxaban/aspirin arm hazard ratios to estimate the potential impact on the COMPASS-eligible cohort. Among 362 797 patients with coronary artery disease or peripheral artery disease, 38% were deemed eligible, 47% ineligible, and 15% indeterminate. Among eligible patients, a greater number of risk factors was associated with higher rates of cardiovascular outcomes, whereas bleeding rates increased minimally. Over 5 years, applying COMPASS treatment effects to eligible patients resulted in a 2.4% absolute risk reduction of major adverse cardiovascular events and a number needed to treat of 42, and a 1.3% absolute risk increase of major bleeding and number needed to harm (NNH) of 77. Those with at least 2 risk factors had a 3.0% absolute risk reduction of major adverse cardiovascular events (number needed to treat =34) and a 1.6% absolute risk increase of major bleeding (number needed to harm =61). Conclusions Implementation of very-low-dose rivaroxaban therapy would potentially impact ≈$$ \approx $$2 in 5 patients with atherosclerotic disease in Ontario. Eligible individuals with ≥$$ \ge $$2 comorbidities represent a high-risk subgroup that may derive the greatest benefit-to-risk ratio. Selection of patients with high-risk predisposing factors appears appropriate in routine practice.

Loss of ATRX promotes aggressive features of osteosarcoma with increased NF-κB signaling and integrin binding.

Osteosarcoma (OS) is a lethal disease with few known targeted therapies. Here, we show that decreased ATRX expression is associated with more aggressive tumor cell phenotypes, including increased growth, migration, invasion, and metastasis. These phenotypic changes correspond with activation of NF-κB signaling, extracellular matrix remodeling, increased integrin αvß3 expression, and ETS family transcription factor binding. Here, we characterize these changes in vitro, in vivo, and in a data set of human OS patients. This increased aggression substantially sensitizes ATRX-deficient OS cells to integrin signaling inhibition. Thus, ATRX plays an important tumor-suppression role in OS, and loss of function of this gene may underlie new therapeutic vulnerabilities. The relationship between ATRX expression and integrin binding, NF-κB activation, and ETS family transcription factor binding has not been described in previous studies and may impact the pathophysiology of other diseases with ATRX loss, including other cancers and the ATR-X α thalassemia intellectual disability syndrome.

Treatment of Chondroblastoma with Denosumab: A Case Report with a Correlative Analysis of Effect on the RANK Signaling Pathway.

CASE: A 15-year-old boy with chondroblastoma of the right hemipelvis presented with significant periacetabular bone destruction. Neoadjuvant denosumab treatment facilitated initial joint preserving surgery. Unfortunately, he experienced 2 local recurrences and underwent wide surgical resection 2 years after his initial diagnosis. CONCLUSION: Inhibition of the receptor activator of NF-κB (RANK)/RANK ligand (RANK-L) pathway with denosumab has been used neoadjuvantly for the treatment of giant cell tumor of bone, but its role in the treatment of chondroblastoma is less understood. This patient's clinical response and effect on cellular RANK/RANK-L activity support the consideration of denosumab in the treatment algorithm for other osteolytic bone tumors such as chondroblastoma.

A Comparative Oncology Drug Discovery Pipeline to Identify and Validate New Treatments for Osteosarcoma.

BACKGROUND: Osteosarcoma is a rare but aggressive bone cancer that occurs primarily in children. Like other rare cancers, treatment advances for osteosarcoma have stagnated, with little improvement in survival for the past several decades. Developing new treatments has been hampered by extensive genomic heterogeneity and limited access to patient samples to study the biology of this complex disease. METHODS: To overcome these barriers, we combined the power of comparative oncology with patient-derived models of cancer and high-throughput chemical screens in a cross-species drug discovery pipeline. RESULTS: Coupling in vitro high-throughput drug screens on low-passage and established cell lines with in vivo validation in patient-derived xenografts we identify the proteasome and CRM1 nuclear export pathways as therapeutic sensitivities in osteosarcoma, with dual inhibition of these pathways inducing synergistic cytotoxicity. CONCLUSIONS: These collective efforts provide an experimental framework and set of new tools for osteosarcoma and other rare cancers to identify and study new therapeutic vulnerabilities.

From the Clinic to the Bench and Back Again in One Dog Year: How a Cross-Species Pipeline to Identify New Treatments for Sarcoma Illuminates the Path Forward in Precision Medicine.

Cancer drug discovery is an inefficient process, with more than 90% of newly-discovered therapies failing to gain regulatory approval. Patient-derived models of cancer offer a promising new approach to identify new treatments; however, for rare cancers, such as sarcomas, access to patient samples is limited, which precludes development of patient-derived models. To address the limited access to patient samples, we have turned to pet dogs with naturally-occurring sarcomas. Although sarcomas make up <1% of all human cancers, sarcomas represent 15% of cancers in dogs. Because dogs have similar immune systems, an accelerated pace of cancer progression, and a shared environment with humans, studying pet dogs with cancer is ideal for bridging gaps between mouse models and human cancers. Here, we present our cross-species personalized medicine pipeline to identify new therapies for sarcomas. We explore this process through the focused study of a pet dog, Teddy, who presented with six synchronous leiomyosarcomas. Using our pipeline we identified proteasome inhibitors as a potential therapy for Teddy. Teddy was treated with bortezomib and showed a varied response across tumors. Whole exome sequencing revealed substantial genetic heterogeneity across Teddy's recurrent tumors and metastases, suggesting that intra-patient heterogeneity and tumoral adaptation were responsible for the heterogeneous clinical response. Ubiquitin proteomics coupled with exome sequencing revealed multiple candidate driver mutations in proteins related to the proteasome pathway. Together, our results demonstrate how the comparative study of canine sarcomas offers important insights into the development of personalized medicine approaches that can lead to new treatments for sarcomas in both humans and canines.

Improving Cancer Drug Discovery by Studying Cancer across the Tree of Life.

Despite a considerable expenditure of time and resources and significant advances in experimental models of disease, cancer research continues to suffer from extremely low success rates in translating preclinical discoveries into clinical practice. The continued failure of cancer drug development, particularly late in the course of human testing, not only impacts patient outcomes, but also drives up the cost for those therapies that do succeed. It is clear that a paradigm shift is necessary if improvements in this process are to occur. One promising direction for increasing translational success is comparative oncology-the study of cancer across species, often involving veterinary patients that develop naturally-occurring cancers. Comparative oncology leverages the power of cross-species analyses to understand the fundamental drivers of cancer protective mechanisms, as well as factors contributing to cancer initiation and progression. Clinical trials in veterinary patients with cancer provide an opportunity to evaluate novel therapeutics in a setting that recapitulates many of the key features of human cancers, including genomic aberrations that underly tumor development, response and resistance to treatment, and the presence of comorbidities that can affect outcomes. With a concerted effort from basic scientists, human physicians and veterinarians, comparative oncology has the potential to enhance the cost-effectiveness and efficiency of pipelines for cancer drug discovery and other cancer treatments.

An Integrative Systems Biology and Experimental Approach Identifies Convergence of Epithelial Plasticity, Metabolism, and Autophagy to Promote Chemoresistance.

The evolution of therapeutic resistance is a major cause of death for cancer patients. The development of therapy resistance is shaped by the ecological dynamics within the tumor microenvironment and the selective pressure of the host immune system. These selective forces often lead to evolutionary convergence on pathways or hallmarks that drive progression. Thus, a deeper understanding of the evolutionary convergences that occur could reveal vulnerabilities to treat therapy-resistant cancer. To this end, we combined phylogenetic clustering, systems biology analyses, and molecular experimentation to identify convergences in gene expression data onto common signaling pathways. We applied these methods to derive new insights about the networks at play during transforming growth factor-ß (TGF-ß)-mediated epithelial⁻mesenchymal transition in lung cancer. Phylogenetic analyses of gene expression data from TGF-ß-treated cells revealed convergence of cells toward amine metabolic pathways and autophagy during TGF-ß treatment. Knockdown of the autophagy regulatory, ATG16L1, re-sensitized lung cancer cells to cancer therapies following TGF-ß-induced resistance, implicating autophagy as a TGF-ß-mediated chemoresistance mechanism. In addition, high ATG16L expression was found to be a poor prognostic marker in multiple cancer types. These analyses reveal the usefulness of combining evolutionary and systems biology methods with experimental validation to illuminate new therapeutic vulnerabilities for cancer.

Hybrid epithelial/mesenchymal phenotypes promote metastasis and therapy resistance across carcinomas.

Cancer metastasis and therapy resistance are the major unsolved clinical challenges, and account for nearly all cancer-related deaths. Both metastasis and therapy resistance are fueled by epithelial plasticity, the reversible phenotypic transitions between epithelial and mesenchymal phenotypes, including epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET). EMT and MET have been largely considered as binary processes, where cells detach from the primary tumor as individual units with many, if not all, traits of a mesenchymal cell (EMT) and then convert back to being epithelial (MET). However, recent studies have demonstrated that cells can metastasize in ways alternative to traditional EMT paradigm; for example, they can detach as clusters, and/or occupy one or more stable hybrid epithelial/mesenchymal (E/M) phenotypes that can be the end point of a transition. Such hybrid E/M cells can integrate various epithelial and mesenchymal traits and markers, facilitating collective cell migration. Furthermore, these hybrid E/M cells may possess higher tumor-initiation and metastatic potential as compared to cells on either end of the EMT spectrum. Here, we review in silico, in vitro, in vivo and clinical evidence for the existence of one or more hybrid E/M phenotype(s) in multiple carcinomas, and discuss their implications in tumor-initiation, tumor relapse, therapy resistance, and metastasis. Together, these studies drive the emerging notion that cells in a hybrid E/M phenotype may occupy 'metastatic sweet spot' in multiple subtypes of carcinomas, and pathways linked to this (these) hybrid E/M state(s) may be relevant as prognostic biomarkers as well as a promising therapeutic targets.