Large-Scale Topological Changes Restrain Malignant Progression in Colorectal Cancer.

Widespread changes to DNA methylation and chromatin are well documented in cancer, but the fate of higher-order chromosomal structure remains obscure. Here we integrated topological maps for colon tumors and normal colons with epigenetic, transcriptional, and imaging data to characterize alterations to chromatin loops, topologically associated domains, and large-scale compartments. We found that spatial partitioning of the open and closed genome compartments is profoundly compromised in tumors. This reorganization is accompanied by compartment-specific hypomethylation and chromatin changes. Additionally, we identify a compartment at the interface between the canonical A and B compartments that is reorganized in tumors. Remarkably, similar shifts were evident in non-malignant cells that have accumulated excess divisions. Our analyses suggest that these topological changes repress stemness and invasion programs while inducing anti-tumor immunity genes and may therefore restrain malignant progression. Our findings call into question the conventional view that tumor-associated epigenomic alterations are primarily oncogenic.

Cancer-Specific Retargeting of BAF Complexes by a Prion-like Domain.

Alterations in transcriptional regulators can orchestrate oncogenic gene expression programs in cancer. Here, we show that the BRG1/BRM-associated factor (BAF) chromatin remodeling complex, which is mutated in over 20% of human tumors, interacts with EWSR1, a member of a family of proteins with prion-like domains (PrLD) that are frequent partners in oncogenic fusions with transcription factors. In Ewing sarcoma, we find that the BAF complex is recruited by the EWS-FLI1 fusion protein to tumor-specific enhancers and contributes to target gene activation. This process is a neomorphic property of EWS-FLI1 compared to wild-type FLI1 and depends on tyrosine residues that are necessary for phase transitions of the EWSR1 prion-like domain. Furthermore, fusion of short fragments of EWSR1 to FLI1 is sufficient to recapitulate BAF complex retargeting and EWS-FLI1 activities. Our studies thus demonstrate that the physical properties of prion-like domains can retarget critical chromatin regulatory complexes to establish and maintain oncogenic gene expression programs.

In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates.

Gene-editing technologies, which include the CRISPR-Cas nucleases1-3 and CRISPR base editors4,5, have the potential to permanently modify disease-causing genes in patients6. The demonstration of durable editing in target organs of nonhuman primates is a key step before in vivo administration of gene editors to patients in clinical trials. Here we demonstrate that CRISPR base editors that are delivered in vivo using lipid nanoparticles can efficiently and precisely modify disease-related genes in living cynomolgus monkeys (Macaca fascicularis). We observed a near-complete knockdown of PCSK9 in the liver after a single infusion of lipid nanoparticles, with concomitant reductions in blood levels of PCSK9 and low-density lipoprotein cholesterol of approximately 90% and about 60%, respectively; all of these changes remained stable for at least 8 months after a single-dose treatment. In addition to supporting a 'once-and-done' approach to the reduction of low-density lipoprotein cholesterol and the treatment of atherosclerotic cardiovascular disease (the leading cause of death worldwide7), our results provide a proof-of-concept for how CRISPR base editors can be productively applied to make precise single-nucleotide changes in therapeutic target genes in the liver, and potentially in other organs.

Epigenome editing of microsatellite repeats defines tumor-specific enhancer functions and dependencies.

Various types of repetitive sequences are dysregulated in cancer. In Ewing sarcoma, the oncogenic fusion protein EWS-FLI1 induces chromatin features typical of active enhancers at GGAA microsatellite repeats, but the function of these sites has not been directly demonstrated. Here, by combining nascent transcription profiling with epigenome editing, we found that a subset of GGAA microsatellite repeats is transcriptionally active in Ewing sarcoma and that silencing individual repeats abolishes local nascent transcription and leads to markedly reduced expression of putative target genes. Epigenome silencing of these repeat sites does not affect gene expression in unrelated cells, can prevent the induction of gene expression by EWS-FLI1, and, in the case of a GGAA repeat that controls SOX2 expression from a distance of 470 kb, is sufficient to impair the growth of Ewing sarcoma xenografts. Using an experimental approach that is broadly applicable to testing different types of repetitive genomic elements, our study directly demonstrates that specific repeat microsatellites can have critical gene regulation functions in cancer and thus represent tumor-specific vulnerabilities that may be exploited to develop new therapies.

Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors.

CRISPR-Cas base-editor technology enables targeted nucleotide alterations, and is being increasingly used for research and potential therapeutic applications1,2. The most widely used cytosine base editors (CBEs) induce deamination of DNA cytosines using the rat APOBEC1 enzyme, which is targeted by a linked Cas protein-guide RNA complex3,4. Previous studies of the specificity of CBEs have identified off-target DNA edits in mammalian cells5,6. Here we show that a CBE with rat APOBEC1 can cause extensive transcriptome-wide deamination of RNA cytosines in human cells, inducing tens of thousands of C-to-U edits with frequencies ranging from 0.07% to 100% in 38-58% of expressed genes. CBE-induced RNA edits occur in both protein-coding and non-protein-coding sequences and generate missense, nonsense, splice site, and 5' and 3' untranslated region mutations. We engineered two CBE variants bearing mutations in rat APOBEC1 that substantially decreased the number of RNA edits (by more than 390-fold and more than 3,800-fold) in human cells. These variants also showed more precise on-target DNA editing than the wild-type CBE and, for most guide RNAs tested, no substantial reduction in editing efficiency. Finally, we show that an adenine base editor7 can also induce transcriptome-wide RNA edits. These results have implications for the use of base editors in both research and clinical settings, illustrate the feasibility of engineering improved variants with reduced RNA editing activities, and suggest the need to more fully define and characterize the RNA off-target effects of deaminase enzymes in base editor platforms.

Augmenting and directing long-range CRISPR-mediated activation in human cells.

Epigenetic editing is an emerging technology that uses artificial transcription factors (aTFs) to regulate expression of a target gene. Although human genes can be robustly upregulated by targeting aTFs to promoters, the activation induced by directing aTFs to distal transcriptional enhancers is substantially less robust and consistent. Here we show that long-range activation using CRISPR-based aTFs in human cells can be made more efficient and reliable by concurrently targeting an aTF to the target gene promoter. We used this strategy to direct target gene choice for enhancers capable of regulating more than one promoter and to achieve allele-selective activation of human genes by targeting aTFs to single-nucleotide polymorphisms embedded in distally located sequences. Our results broaden the potential applications of the epigenetic editing toolbox for research and therapeutics.

Therapeutic targeting of LCK tyrosine kinase and mTOR signaling in T-cell acute lymphoblastic leukemia.

Relapse and refractory T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis, and new combination therapies are sorely needed. Here, we used an ex vivo high-throughput screening platform to identify drug combinations that kill zebrafish T-ALL and then validated top drug combinations for preclinical efficacy in human disease. This work uncovered potent drug synergies between AKT/mTORC1 (mammalian target of rapamycin complex 1) inhibitors and the general tyrosine kinase inhibitor dasatinib. Importantly, these same drug combinations effectively killed a subset of relapse and dexamethasone-resistant zebrafish T-ALL. Clinical trials are currently underway using the combination of mTORC1 inhibitor temsirolimus and dasatinib in other pediatric cancer indications, leading us to prioritize this therapy for preclinical testing. This combination effectively curbed T-ALL growth in human cell lines and primary human T-ALL and was well tolerated and effective in suppressing leukemia growth in patient-derived xenografts (PDX) grown in mice. Mechanistically, dasatinib inhibited phosphorylation and activation of the lymphocyte-specific protein tyrosine kinase (LCK) to blunt the T-cell receptor (TCR) signaling pathway, and when complexed with mTORC1 inhibition, induced potent T-ALL cell killing through reducing MCL-1 protein expression. In total, our work uncovered unexpected roles for the LCK kinase and its regulation of downstream TCR signaling in suppressing apoptosis and driving continued leukemia growth. Analysis of a wide array of primary human T-ALLs and PDXs grown in mice suggest that combination of temsirolimus and dasatinib treatment will be efficacious for a large fraction of human T-ALLs.

Dementia "e"-consults for Behavioral and Psychological Symptoms of Dementia: Improving access to specialty dementia care for rural Veterans.

OBJECTIVES: This paper characterizes a telephone-based e-consult program designed to assess and treat behavioral and psychological symptoms of dementia (BPSD) for older rural Veterans. METHODS: E-consults required geriatricians to conduct chart review and telephone calls to caregivers to determine behavior triggers, prior management attempts, and medications. Pharmacologic and non-pharmacological recommendations were provided with follow-up calls as needed. RESULTS: Evaluation of 364 Veterans (M age = 80.8, 32% in rural/distal clinics) showed 97% (n = 355) of E-consult interventions included caregiver dementia education to prepare them for managing disease progression and provide non-pharmacological strategies for BPSD. Ninety-four percent (n = 244) of Veterans received medication guidance. A total of 37,504 travel miles was saved, with an average of 108 miles for each Veteran. CONCLUSIONS: Findings support continued implementation of telephone and other virtual modalities of assessing and treating BPSD for older Veterans, thereby increasing access to dementia specialists, especially for rural older adults and their caregivers. A limitation to e-consults is the time needed to provide services compared to the maximum workload credit allowed. CLINICAL IMPLICATIONS: Virtual care improves access to Geriatric specialists and semi-urgent care that otherwise is not available. E-consults are effective in providing primary care providers guidance for diagnosis and management of dementia.

Caregiver Experience of Tele-dementia Care for Older Veterans.

BACKGROUND: For the 5 million persons living with dementia (PLWD) in the USA, telemedicine may improve access to specialty care from their homes. OBJECTIVE: To elicit informal caregiver perceptions of tele-dementia care provided during COVID-19. DESIGN: Qualitative, observational study using grounded theory. PARTICIPANTS: Informal caregivers aged 18 + who cared for an older adult who received tele-dementia services at two major VA healthcare systems participated in 30-60-min semi-structured telephone interviews. INTERVENTIONS: Interviews were designed using Fortney's Access to Care model. MAIN MEASURES: Thirty caregivers (mean age = 67, SD = 12, 87% women) were interviewed. KEY RESULTS: Five major themes were (1) Tele-dementia care avoids routine disruption and pre-visit stress; (2) Transportation barriers to in-person visits include not only travel logistics but navigating the sequelae of dementia and comorbid medical conditions. These include cognitive, behavioral, physical, and emotional challenges such as balance issues, incontinence, and agitation in traffic; (3) Tele-dementia care saves time and money and improves access to specialists; (4) Tele-dementia facilitated communication between caregiver and provider without hindering communication between PLWD and provider; and (5) Caregivers described ideal future dementia care as a combination of virtual and in-person modalities with in-home help, financial and medical support, and dementia-sensitive caregiver access. Caregivers interviewed saved 2.6 h ± 1.5 h (range: 0.5 to 6 h) of travel time. Multiple caregivers described disruption of routines as difficult in PLWD and appreciated the limited preparation and immediate return to routine post telemedicine visit as positives. CONCLUSIONS: Caregivers found tele-dementia care convenient, comfortable, stress reducing, timesaving, and highly satisfactory. Caregivers would prefer a combination of in-person and telemedicine visits, with an opportunity to communicate with providers privately. This intervention prioritizes care for older Veterans with dementia who have high care needs and are at higher risk for hospitalization than their same age counterparts without dementia.

Feasibility, Acceptance, and Initial Evaluation of a Telephone-Based Program Designed to Increase Socialization in Older Veterans.

Loneliness is a public health issue, particularly for older Veterans. To increase older Veterans' access for socialization opportunities, a community-based telephone-delivered activity program was developed, in which Veterans can call in and engage in social activities through telephone. This paper illustrates the feasibility, acceptance, and preliminary outcomes of this program using a mixed-methods design. Thirty-two Veterans enrolled in the program, with 14 attendees who called in to the program at least once. Attendees were more likely to be depressed than those who did not call in at baseline. Program was acceptable with high client satisfaction. Perceived benefits included a structured program with interesting topics to spend time on and the opportunity to socialize, exchange ideas, and connect with other Veterans. Individual challenges (e.g., hearing difficulty) and program-level challenges (e.g., complicated procedures) were reported during qualitative interviews. Among attendees, a significant decrease in loneliness from baseline to 3-months was found but should be interpreted with caution based on the small sample size. While positive findings emerged regarding feasibility, acceptance, preliminary benefits of this program, further refinement is needed to improve future program implementation.

A (fire)cloud-based DNA methylation data preprocessing and quality control platform.

BACKGROUND: Bisulfite sequencing allows base-pair resolution profiling of DNA methylation and has recently been adapted for use in single-cells. Analyzing these data, including making comparisons with existing data, remains challenging due to the scale of the data and differences in preprocessing methods between published datasets. RESULTS: We present a set of preprocessing pipelines for bisulfite sequencing DNA methylation data that include a new R/Bioconductor package, scmeth, for a series of efficient QC analyses of large datasets. The pipelines go from raw data to CpG-level methylation estimates and can be run, with identical results, either on a single computer, in an HPC cluster or on Google Cloud Compute resources. These pipelines are designed to allow users to 1) ensure reproducibility of analyses, 2) achieve scalability to large whole genome datasets with 100 GB+ of raw data per sample and to single-cell datasets with thousands of cells, 3) enable integration and comparison between user-provided data and publicly available data, as all samples can be processed through the same pipeline, and 4) access to best-practice analysis pipelines. Pipelines are provided for whole genome bisulfite sequencing (WGBS), reduced representation bisulfite sequencing (RRBS) and hybrid selection (capture) bisulfite sequencing (HSBS). CONCLUSIONS: The workflows produce data quality metrics, visualization tracks, and aggregated output for further downstream analysis. Optional use of cloud computing resources facilitates analysis of large datasets, and integration with existing methylome profiles. The workflow design principles are applicable to other genomic data types.

Modulation of complex coordinated molecular signaling by 5HT and a cocktail of inhibitors leads to ovarian maturation of Penaeus monodon in captivity.

In aquaculture practices, prawn cultivation holds the major share and Penaeus monodon is the main species cultured. The decline in production of P. monodon is mainly due to the limited availability of domesticated broodstock, which is attributed to its reproductive cycle, controlled by complex coordinated signaling mechanisms. Unilateral eyestalk ablation of domesticated females held in captivity is done to induce ovary development, which has certain disadvantages, including a high rate of mortality. Thus, developing alternative techniques for eyestalk ablation in captive broodstock is necessary to induce maturation of ovary. This study exemplifies the role of 5HT along with a cocktail of inhibitors (U0126, Rp-cAMP, and LY294002) in inducing ovarian maturation. In this study, inhibition of pERK by U0126 inhibited vitellogenesis-inhibiting hormone (VIH), which in turn led to the overexpression of vitellogenin. 5HT induces steroidogenesis (estradiol-17ß) through induction of the gonadotropin-releasing hormone by activating calcium-calmodulin signaling. Steroidogenesis is also aided by synthesis of StAR protein. Estradiol-17ß stimulates the formation of the maturation-promoting factor (MPF) complex by cdc25 activation and Myt1 inactivation. LY294002 aids in keeping cdc25 activated by inhibiting calcium-calmodulin induced phosphorylation of Akt which is a negative regulator of mitogen-activated protein kinases. VIH induced activation of Myt1, through protein kinase A (PKA), was inhibited by Rp-cAMP which inhibits adenylate cyclase, thus stabilizing the activated MPF complex. To conclude, the coordinated effect of inhibitors and 5HT accelerates the development of ovary from previtellogenic to matured oocytes, yielding high quality and quantity larvae compared with eyestalk-ablated P. monodon.

Sequence features and chromatin structure around the genomic regions bound by 119 human transcription factors.

Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) has become the dominant technique for mapping transcription factor (TF) binding regions genome-wide. We performed an integrative analysis centered around 457 ChIP-seq data sets on 119 human TFs generated by the ENCODE Consortium. We identified highly enriched sequence motifs in most data sets, revealing new motifs and validating known ones. The motif sites (TF binding sites) are highly conserved evolutionarily and show distinct footprints upon DNase I digestion. We frequently detected secondary motifs in addition to the canonical motifs of the TFs, indicating tethered binding and cobinding between multiple TFs. We observed significant position and orientation preferences between many cobinding TFs. Genes specifically expressed in a cell line are often associated with a greater occurrence of nearby TF binding in that cell line. We observed cell-line-specific secondary motifs that mediate the binding of the histone deacetylase HDAC2 and the enhancer-binding protein EP300. TF binding sites are located in GC-rich, nucleosome-depleted, and DNase I sensitive regions, flanked by well-positioned nucleosomes, and many of these features show cell type specificity. The GC-richness may be beneficial for regulating TF binding because, when unoccupied by a TF, these regions are occupied by nucleosomes in vivo. We present the results of our analysis in a TF-centric web repository Factorbook (http://factorbook.org) and will continually update this repository as more ENCODE data are generated.

Factorbook.org: a Wiki-based database for transcription factor-binding data generated by the ENCODE consortium.

The Encyclopedia of DNA Elements (ENCODE) consortium aims to identify all functional elements in the human genome including transcripts, transcriptional regulatory regions, along with their chromatin states and DNA methylation patterns. The ENCODE project generates data utilizing a variety of techniques that can enrich for regulatory regions, such as chromatin immunoprecipitation (ChIP), micrococcal nuclease (MNase) digestion and DNase I digestion, followed by deeply sequencing the resulting DNA. As part of the ENCODE project, we have developed a Web-accessible repository accessible at http://factorbook.org. In Wiki format, factorbook is a transcription factor (TF)-centric repository of all ENCODE ChIP-seq datasets on TF-binding regions, as well as the rich analysis results of these data. In the first release, factorbook contains 457 ChIP-seq datasets on 119 TFs in a number of human cell lines, the average profiles of histone modifications and nucleosome positioning around the TF-binding regions, sequence motifs enriched in the regions and the distance and orientation preferences between motif sites.

GalNAc-Lipid nanoparticles enable non-LDLR dependent hepatic delivery of a CRISPR base editing therapy.

Lipid nanoparticles have demonstrated utility in hepatic delivery of a range of therapeutic modalities and typically deliver their cargo via low-density lipoprotein receptor-mediated endocytosis. For patients lacking sufficient low-density lipoprotein receptor activity, such as those with homozygous familial hypercholesterolemia, an alternate strategy is needed. Here we show the use of structure-guided rational design in a series of mouse and non-human primate studies to optimize a GalNAc-Lipid nanoparticle that allows for low-density lipoprotein receptor independent delivery. In low-density lipoprotein receptor-deficient non-human primates administered a CRISPR base editing therapy targeting the ANGPTL3 gene, the introduction of an optimized GalNAc-based asialoglycoprotein receptor ligand to the nanoparticle surface increased liver editing from 5% to 61% with minimal editing in nontargeted tissues. Similar editing was noted in wild-type monkeys, with durable blood ANGPTL3 protein reduction up to 89% six months post dosing. These results suggest that GalNAc-Lipid nanoparticles may effectively deliver to both patients with intact low-density lipoprotein receptor activity as well as those afflicted by homozygous familial hypercholesterolemia.

Highly connected 3D chromatin networks established by an oncogenic fusion protein shape tumor cell identity.

Cell fate transitions observed in embryonic development involve changes in three-dimensional genomic organization that provide proper lineage specification. Whether similar events occur within tumor cells and contribute to cancer evolution remains largely unexplored. We modeled this process in the pediatric cancer Ewing sarcoma and investigated high-resolution looping and large-scale nuclear conformation changes associated with the oncogenic fusion protein EWS-FLI1. We show that chromatin interactions in tumor cells are dominated by highly connected looping hubs centered on EWS-FLI1 binding sites, which directly control the activity of linked enhancers and promoters to establish oncogenic expression programs. Conversely, EWS-FLI1 depletion led to the disassembly of these looping networks and a widespread nuclear reorganization through the establishment of new looping patterns and large-scale compartment configuration matching those observed in mesenchymal stem cells, a candidate Ewing sarcoma progenitor. Our data demonstrate that major architectural features of nuclear organization in cancer cells can depend on single oncogenes and are readily reversed to reestablish latent differentiation programs.

Delivering Virtual Care to Patients with Cognitive Impairment within the Veterans Health Administration: Multi-level Barriers and Solutions.

Older patients with cognitive impairment, including dementia, may benefit from virtual care that increases access to geriatric specialties. Here, we identify clinician-level strategies to address the numerous barriers that reduce utilization of virtual services. We describe two innovative programs in the Veterans Health Administration that deliver geriatric medicine and geriatric psychiatry services virtually. This commentary outlines concrete strategies addressing identified barriers, including technology access, digital literacy, and ambivalence and communication challenges during video visits. Two virtual care programs (tele-geriatric psychiatry consultation; tele-dementia care) that address complex medical and mental health issues in older adults with cognitive impairment are described. The Consolidated Framework for Implementation Research (CFIR) is used to categorize the clinician-level strategies and program elements as they relate to the implementation domains and constructs. Clinicians can use education strategies prior to and during virtual care visits to facilitate access to video, optimize the virtual experience, and promote information retention. These strategies rely on aspects of the inner setting, outer setting, and characteristics of individuals. The two virtual programs vary in their intervention characteristics and the inner setting, yet both programs share similar characteristics of individuals. Key elements contributing to adoption and sustainment of these virtual care programs for patients with cognitive impairment include the relative advantage of virtual care to leverage access to specialists over alternative solutions in each setting. Other factors to consider include the importance of communication, program champions, and the role of the Veterans Health Administration.

Mutant IL7R collaborates with MYC to induce T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive pediatric cancer. Amongst the wide array of driver mutations, 10% of T-ALL patients display gain-of-function mutations in the IL-7 receptor α chain (IL-7Rα, encoded by IL7R), which occur in different molecular subtypes of this disease. However, it is still unclear whether IL-7R mutational activation is sufficient to transform T-cell precursors. Also, which genes cooperate with IL7R to drive leukemogenesis remain poorly defined. Here, we demonstrate that mutant IL7R alone is capable of inducing T-ALL with long-latency in stable transgenic zebrafish and transformation is associated with MYC transcriptional activation. Additionally, we find that mutant IL7R collaborates with Myc to induce early onset T-ALL in transgenic zebrafish, supporting a model where these pathways collaborate to drive leukemogenesis. T-ALLs co-expressing mutant IL7R and Myc activate STAT5 and AKT pathways, harbor reduced numbers of apoptotic cells and remake tumors in transplanted zebrafish faster than T-ALLs expressing Myc alone. Moreover, limiting-dilution cell transplantation experiments reveal that activated IL-7R signaling increases the overall frequency of leukemia propagating cells. Our work highlights a synergy between mutant IL7R and Myc in inducing T-ALL and demonstrates that mutant IL7R enriches for leukemia propagating potential.

CD4+ T helper 2 cells suppress breast cancer by inducing terminal differentiation.

Cancer immunology research is largely focused on the role of cytotoxic immune responses against advanced cancers. Herein, we demonstrate that CD4+ T helper (Th2) cells directly block spontaneous breast carcinogenesis by inducing the terminal differentiation of the cancer cells. Th2 cell immunity, stimulated by thymic stromal lymphopoietin, caused the epigenetic reprogramming of the tumor cells, activating mammary gland differentiation and suppressing epithelial-mesenchymal transition. Th2 polarization was required for this tumor antigen-specific immunity, which persisted in the absence of CD8+ T and B cells. Th2 cells directly blocked breast carcinogenesis by secreting IL-3, IL-5, and GM-CSF, which signaled to their common receptor expressed on breast tumor cells. Importantly, Th2 cell immunity permanently reverted high-grade breast tumors into low-grade, fibrocystic-like structures. Our findings reveal a critical role for CD4+ Th2 cells in immunity against breast cancer, which is mediated by terminal differentiation as a distinct effector mechanism for cancer immunoprevention and therapy.