EWS-WT1 fusion isoforms establish oncogenic programs and therapeutic vulnerabilities in desmoplastic small round cell tumors.

EWS fusion oncoproteins underlie several human malignancies including Desmoplastic Small Round Cell Tumor (DSRCT), an aggressive cancer driven by EWS-WT1 fusion proteins. Here we combine chromatin occupancy and 3D profiles to identify EWS-WT1-dependent gene regulation networks and target genes. We show that EWS-WT1 is a powerful chromatin activator controlling an oncogenic gene expression program that characterizes primary tumors. Similar to wild type WT1, EWS-WT1 has two isoforms that differ in their DNA binding domain and we find that they have distinct DNA binding profiles and are both required to generate viable tumors that resemble primary DSRCT. Finally, we identify candidate EWS-WT1 target genes with potential therapeutic implications, including CCND1, whose inhibition by the clinically-approved drug Palbociclib leads to marked tumor burden decrease in DSRCT PDXs in vivo. Taken together, our studies identify gene regulation programs and therapeutic vulnerabilities in DSRCT and provide a mechanistic understanding of the complex oncogenic activity of EWS-WT1.

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.

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.

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.

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.

Genome-wide functional perturbation of human microsatellite repeats using engineered zinc finger transcription factors.

Repeat elements can be dysregulated at a genome-wide scale in human diseases. For example, in Ewing sarcoma, hundreds of inert GGAA repeats can be converted into active enhancers when bound by EWS-FLI1. Here we show that fusions between EWS and GGAA-repeat-targeted engineered zinc finger arrays (ZFAs) can function at least as efficiently as EWS-FLI1 for converting hundreds of GGAA repeats into active enhancers in a Ewing sarcoma precursor cell model. Furthermore, a fusion of a KRAB domain to a ZFA can silence GGAA microsatellite enhancers genome wide in Ewing sarcoma cells, thereby reducing expression of EWS-FLI1-activated genes. Remarkably, this KRAB-ZFA fusion showed selective toxicity against Ewing sarcoma cells compared with non-Ewing cancer cells, consistent with its Ewing sarcoma-specific impact on the transcriptome. These findings demonstrate the value of ZFAs for functional annotation of repeats and illustrate how aberrant microsatellite activities might be regulated for potential therapeutic applications.

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.

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.

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.

EWSR1-ATF1 dependent 3D connectivity regulates oncogenic and differentiation programs in Clear Cell Sarcoma.

Oncogenic fusion proteins generated by chromosomal translocations play major roles in cancer. Among them, fusions between EWSR1 and transcription factors generate oncogenes with powerful chromatin regulatory activities, capable of establishing complex gene expression programs in permissive precursor cells. Here we define the epigenetic and 3D connectivity landscape of Clear Cell Sarcoma, an aggressive cancer driven by the EWSR1-ATF1 fusion gene. We find that EWSR1-ATF1 displays a distinct DNA binding pattern that requires the EWSR1 domain and promotes ATF1 retargeting to new distal sites, leading to chromatin activation and the establishment of a 3D network that controls oncogenic and differentiation signatures observed in primary CCS tumors. Conversely, EWSR1-ATF1 depletion results in a marked reconfiguration of 3D connectivity, including the emergence of regulatory circuits that promote neural crest-related developmental programs. Taken together, our study elucidates the epigenetic mechanisms utilized by EWSR1-ATF1 to establish regulatory networks in CCS, and points to precursor cells in the neural crest lineage as candidate cells of origin for these tumors.

Single-cell imaging of T cell immunotherapy responses in vivo.

T cell immunotherapies have revolutionized treatment for a subset of cancers. Yet, a major hurdle has been the lack of facile and predicative preclinical animal models that permit dynamic visualization of T cell immune responses at single-cell resolution in vivo. Here, optically clear immunocompromised zebrafish were engrafted with fluorescent-labeled human cancers along with chimeric antigen receptor T (CAR T) cells, bispecific T cell engagers (BiTEs), and antibody peptide epitope conjugates (APECs), allowing real-time single-cell visualization of T cell-based immunotherapies in vivo. This work uncovered important differences in the kinetics of T cell infiltration, tumor cell engagement, and killing between these immunotherapies and established early endpoint analysis to predict therapy responses. We also established EGFR-targeted immunotherapies as a powerful approach to kill rhabdomyosarcoma muscle cancers, providing strong preclinical rationale for assessing a wider array of T cell immunotherapies in this disease.

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.

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.

The deacylase SIRT5 supports melanoma viability by influencing chromatin dynamics.

Cutaneous melanoma remains the most lethal skin cancer, and ranks third among all malignancies in terms of years of life lost. Despite the advent of immune checkpoint and targeted therapies, only roughly half of patients with advanced melanoma achieve a durable remission. Sirtuin 5 (SIRT5) is a member of the sirtuin family of protein deacylases that regulates metabolism and other biological processes. Germline Sirt5 deficiency is associated with mild phenotypes in mice. Here we showed that SIRT5 was required for proliferation and survival across all cutaneous melanoma genotypes tested, as well as uveal melanoma, a genetically distinct melanoma subtype that arises in the eye and is incurable once metastatic. Likewise, SIRT5 was required for efficient tumor formation by melanoma xenografts and in an autochthonous mouse Braf Pten-driven melanoma model. Via metabolite and transcriptomic analyses, we found that SIRT5 was required to maintain histone acetylation and methylation levels in melanoma cells, thereby promoting proper gene expression. SIRT5-dependent genes notably included MITF, a key lineage-specific survival oncogene in melanoma, and the c-MYC proto-oncogene. SIRT5 may represent a druggable genotype-independent addiction in melanoma.

Converting a Geriatrics Clinic to Virtual Visits during COVID-19: A Case Study.

BACKGROUND: To characterize the experience of converting a geriatrics clinic to telehealth visits in early stages of a pandemic. DESIGN: An organizational case study with mixed methods evaluation from the first 8 weeks of converting a geriatrics clinic from in-person visits to video and telephone visits. SETTING: Veteran's Health Administration in Northern California Participants Community-dwelling older Veterans receiving care at VA Palo Alto Geriatrics clinic. Veterans had a mean age of 85.7 (SD = 6.8) and 72.1% had cognitive impairment. INTERVENTION: Veterans with face-to-face appointments were converted to video or telephone visits to mitigate exposure to community spread of COVID-19. MEASUREMENTS: Thirty-two patient evaluations and 80 clinician feedback evaluations were completed. This provided information on satisfaction, care access during pandemic, and travel and time savings. RESULTS: Of the 62 scheduled appointments, 43 virtual visits (69.4%) were conducted. Twenty-six (60.5%) visits were conducted via video, 17 (39.5%) by telephone. Virtual visits saved patients an average of 118.6 minutes each. Patients and providers had similar, positive perceptions about telehealth to in-person visit comparison, limiting exposure, and visit satisfaction. After the telehealth appointment, patients indicated greater comfort with using virtual visits in the future. Thirty-one evaluations included comments for qualitative analysis. We identified 3 main themes of technology set-up and usability, satisfaction with visit, and clinical assessment and communication. CONCLUSION: During a pandemic that has limited the ability to safely conduct inperson services, virtual formats offer a feasible and acceptable alternative for clinically-complex older patients. Despite potential barriers and additional effort required for telehealth visits, patients expressed willingness to utilize this format. Patients and providers reported high satisfaction, particularly with the ability to access care similar to in-person while staying safe. Investing in telehealth services during a pandemic ensures that vulnerable older patients can access care while maintaining social distancing, an important safety measure.

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.

CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells.

CRISPR-guided DNA cytosine and adenine base editors are widely used for many applications1-4 but primarily create DNA base transitions (that is, pyrimidine-to-pyrimidine or purine-to-purine). Here we describe the engineering of two base editor architectures that can efficiently induce targeted C-to-G base transversions, with reduced levels of unwanted C-to-W (W = A or T) and indel mutations. One of these C-to-G base editors (CGBE1), consists of an RNA-guided Cas9 nickase, an Escherichia coli-derived uracil DNA N-glycosylase (eUNG) and a rat APOBEC1 cytidine deaminase variant (R33A) previously shown to have reduced off-target RNA and DNA editing activities5,6. We show that CGBE1 can efficiently induce C-to-G edits, particularly in AT-rich sequence contexts in human cells. We also removed the eUNG domain to yield miniCGBE1, which reduced indel frequencies but only modestly decreased editing efficiency. CGBE1 and miniCGBE1 enable C-to-G edits and will serve as a basis for optimizing C-to-G base editors for research and therapeutic applications.

The chromatin landscape of primary synovial sarcoma organoids is linked to specific epigenetic mechanisms and dependencies.

Synovial sarcoma (SyS) is an aggressive mesenchymal malignancy invariably associated with the chromosomal translocation t(X:18; p11:q11), which results in the in-frame fusion of the BAF complex gene SS18 to one of three SSX genes. Fusion of SS18 to SSX generates an aberrant transcriptional regulator, which, in permissive cells, drives tumor development by initiating major chromatin remodeling events that disrupt the balance between BAF-mediated gene activation and polycomb-dependent repression. Here, we developed SyS organoids and performed genome-wide epigenomic profiling of these models and mesenchymal precursors to define SyS-specific chromatin remodeling mechanisms and dependencies. We show that SS18-SSX induces broad BAF domains at its binding sites, which oppose polycomb repressor complex (PRC) 2 activity, while facilitating recruitment of a non-canonical (nc)PRC1 variant. Along with the uncoupling of polycomb complexes, we observed H3K27me3 eviction, H2AK119ub deposition and the establishment of de novo active regulatory elements that drive SyS identity. These alterations are completely reversible upon SS18-SSX depletion and are associated with vulnerability to USP7 loss, a core member of ncPRC1.1. Using the power of primary tumor organoids, our work helps define the mechanisms of epigenetic dysregulation on which SyS cells are dependent.