The three-dimensional structure of the EBV genome plays a crucial role in regulating viral gene expression in EBVaGC.

Epstein-Barr virus (EBV) establishes lifelong asymptomatic infection by replication of its chromatinized episomes with the host genome. EBV exhibits different latency-associated transcriptional repertoires, each with distinct three-dimensional structures. CTCF, Cohesin and PARP1 are involved in maintaining viral latency and establishing episome architecture. Epstein-Barr virus-associated gastric cancer (EBVaGC) represents 1.3-30.9% of all gastric cancers globally. EBV-positive gastric cancers exhibit an intermediate viral transcription profile known as 'Latency II', expressing specific viral genes and noncoding RNAs. In this study, we investigated the impact of PARP1 inhibition on CTCF/Cohesin binding in Type II latency. We observed destabilization of the binding of both factors, leading to a disrupted three-dimensional architecture of the episomes and an altered viral gene expression. Despite sharing the same CTCF binding profile, Type I, II and III latencies exhibit different 3D structures that correlate with variations in viral gene expression. Additionally, our analysis of H3K27ac-enriched interactions revealed differences between Type II latency episomes and a link to cellular transformation through docking of the EBV genome at specific sites of the Human genome, thus promoting oncogene expression. Overall, this work provides insights into the role of PARP1 in maintaining active latency and novel mechanisms of EBV-induced cellular transformation.

Video Intervention and Goals-of-Care Documentation in Hospitalized Older Adults: The VIDEO-PCE Randomized Clinical Trial.

Importance: Despite the benefits of goals-of-care (GOC) communication, many hospitalized individuals never communicate their goals or preferences to clinicians. Objective: To assess whether a GOC video intervention delivered by palliative care educators (PCEs) increased the rate of GOC documentation. Design, Setting, and Participants: This pragmatic, stepped-wedge cluster randomized clinical trial included patients aged 65 years or older admitted to 1 of 14 units at 2 urban hospitals in New York and Boston from July 1, 2021, to October 31, 2022. Intervention: The intervention involved PCEs (social workers and nurses trained in GOC communication) facilitating GOC conversations with patients and/or their decision-makers using a library of brief, certified video decision aids available in 29 languages. Patients in the control period received usual care. Main Outcome and Measures: The primary outcome was GOC documentation, which included any documentation of a goals conversation, limitation of life-sustaining treatment, palliative care, hospice, or time-limited trials and was obtained by natural language processing. Results: A total of 10 802 patients (mean [SD] age, 78 [8] years; 51.6% male) were admitted to 1 of 14 hospital units. Goals-of-care documentation during the intervention phase occurred among 3744 of 6023 patients (62.2%) compared with 2396 of 4779 patients (50.1%) in the usual care phase (P < .001). Proportions of documented GOC discussions for Black or African American individuals (865 of 1376 [62.9%] vs 596 of 1125 [53.0%]), Hispanic or Latino individuals (311 of 548 [56.8%] vs 218 of 451 [48.3%]), non-English speakers (586 of 1059 [55.3%] vs 405 of 863 [46.9%]), and people living with Alzheimer disease and related dementias (520 of 681 [76.4%] vs 355 of 570 [62.3%]) were greater during the intervention phase compared with the usual care phase. Conclusions and Relevance: In this stepped-wedge cluster randomized clinical trial of older adults, a GOC video intervention delivered by PCEs resulted in higher rates of GOC documentation compared with usual care, including among Black or African American individuals, Hispanic or Latino individuals, non-English speakers, and people living with Alzheimer disease and related dementias. The findings suggest that this form of patient-centered care delivery may be a beneficial decision support tool. Trial Registration: ClinicalTrials.gov Identifier: NCT04857060.

Decitabine disrupts EBV genomic epiallele DNA methylation patterns around CTCF binding sites to increase chromatin accessibility and lytic transcription in gastric cancer.

IMPORTANCE: Epstein-Barr virus (EBV) latency is controlled by epigenetic silencing by DNA methylation [5-methyl cytosine (5mC)], histone modifications, and chromatin looping. However, how they dictate the transcriptional program in EBV-associated gastric cancers remains incompletely understood. EBV-associated gastric cancer displays a 5mC hypermethylated phenotype. A potential treatment for this cancer subtype is the DNA hypomethylating agent, which induces EBV lytic reactivation and targets hypermethylation of the cellular DNA. In this study, we identified a heterogeneous pool of EBV epialleles within two tumor-derived gastric cancer cell lines that are disrupted with a hypomethylating agent. Stochastic DNA methylation patterning at critical regulatory regions may be an underlying mechanism for spontaneous reactivation. Our results highlight the critical role of epigenetic modulation on EBV latency and life cycle, which is maintained through the interaction between 5mC and the host protein CCCTC-binding factor.

PARP1 Inhibition Halts EBV+ Lymphoma Progression by Disrupting the EBNA2/MYC Axis.

PARP1 has been shown to regulate EBV latency. However, the therapeutic effect of PARP1 inhibitors on EBV+ lymphomagenesis has not yet been explored. Here, we show that PARPi BMN-673 has a potent anti-tumor effect on EBV-driven LCL in a mouse xenograft model. We found that PARP1 inhibition induces a dramatic transcriptional reprogramming of LCLs driven largely by the reduction of the MYC oncogene expression and dysregulation of MYC targets, both in vivo and in vitro. PARP1 inhibition also reduced the expression of viral oncoprotein EBNA2, which we previously demonstrated depends on PARP1 for activation of MYC. Further, we show that PARP1 inhibition blocks the chromatin association of MYC, EBNA2, and tumor suppressor p53. Overall, our study strengthens the central role of PARP1 in EBV malignant transformation and identifies the EBNA2/MYC pathway as a target of PARP1 inhibitors and its utility for the treatment of EBNA2-driven EBV-associated cancers.

Evaluation of an Emergency Department-based Palliative Care Extender Program on Hospital and Patient Outcomes.

BACKGROUND: Boston Medical Center (BMC), a safety-net hospital, treated a substantial portion of the Boston cohort that was sick with COVID-19. Unfortunately, these patients experienced high rates of morbidity and mortality given the significant health disparities that many of BMC's patients face. Boston Medical Center launched a palliative care extender program to help address the needs of critically ill ED patients under crisis conditions. In this program evaluation our goal was to assess outcomes between those who received palliative care in the emergency department (ED) vs those who received palliative care as an inpatient or were admitted to an intensive care unit (ICU). METHODS: We used a matched retrospective cohort study design to assess the difference in outcomes between the two groups. RESULTS: A total of 82 patients received palliative care services in the ED, and 317 patients received palliative care services as an inpatient. After controlling for demographics, patients who received palliative care services in the ED were less likely to have a change in level of care (P<0.001) or be admitted to an ICU (P<0.001). Cases had an average length of stay of 5.2 days compared to controls who stayed 9.9 days (P<0.001). CONCLUSION: Within a busy ED environment, initiating palliative care discussions by ED staff can be challenging. This study demonstrates that consulting palliative care specialists early in the course of the patient's ED stay can benefit patients and families and improve resource utilization.

Three-Dimensional Chromatin Structure of the EBV Genome: A Crucial Factor in Viral Infection.

Epstein-Barr Virus (EBV) is a human gamma-herpesvirus that is widespread worldwide. To this day, about 200,000 cancer cases per year are attributed to EBV infection. EBV is capable of infecting both B cells and epithelial cells. Upon entry, viral DNA reaches the nucleus and undergoes a process of circularization and chromatinization and establishes a latent lifelong infection in host cells. There are different types of latency all characterized by different expressions of latent viral genes correlated with a different three-dimensional architecture of the viral genome. There are multiple factors involved in the regulation and maintenance of this three-dimensional organization, such as CTCF, PARP1, MYC and Nuclear Lamina, emphasizing its central role in latency maintenance.

Human Cytomegalovirus Utilizes Multiple Viral Proteins to Regulate the Basement Membrane Protein Nidogen 1.

Nidogen 1 (NID1) is an important basement membrane protein secreted by many cell types. We previously found that human cytomegalovirus (HCMV) infection rapidly induced chromosome 1 breaks and that the basement membrane protein NID1, encoded near the 1q42 break site, was downregulated. We have now determined that the specific breaks in and of themselves did not regulate NID1, rather interactions between several viral proteins and the cellular machinery and DNA regulated NID1. We screened a battery of viral proteins present by 24 hours postinfection (hpi) when regulation was induced, including components of the incoming virion and immediate early (IE) proteins. Adenovirus (Ad) delivery of the tegument proteins pp71 and UL35 and the IE protein IE1 influenced steady-state (ss) NID1 levels. IE1's mechanism of regulation was unclear, while UL35 influenced proteasomal regulation of ss NID1. Real-time quantitative PCR (RT-qPCR) experiments determined that pp71 downregulated NID1 transcription. Surprisingly, WF28-71, a fibroblast clone that expresses minute quantities of pp71, suppressed NID1 transcription as efficiently as HCMV infection, resulting in the near absence of ss NID1. Sequence analysis of the region surrounding the 1q42 break sites and NID1 promoter revealed CCCTC-binding factor (CTCF) binding sites. Chromatin immunoprecipitation experiments determined that pp71 and CTCF were both bound at these two sites during HCMV infection. Expression of pp71 alone replicated this binding. Binding was observed as early as 1 hpi, and colocalization of pp71 and CTCF occurred as quickly as 15 min postinfection (pi) in infected cell nuclei. In fibroblasts where CTCF was knocked down, Adpp71 infection did not decrease NID1 transcription nor ss NID1 protein levels. Our results emphasize another aspect of pp71 activity during infection and identify this viral protein as a key contributor to HCMV's efforts to eliminate NID1. Further, we show, for the first time, direct interaction between pp71 and the cellular genome. IMPORTANCE We have found that human cytomegalovirus (HCMV) utilizes multiple viral proteins in multiple pathways to regulate a ubiquitous cellular basement membrane protein, nidogen-1 (NID1). The extent of the resources and the redundant methods that the virus has evolved to affect this control strongly suggest that its removal provides a life cycle advantage to HCMV. Our discoveries that one of the proteins that HCMV uses to control NID1, pp71, binds directly to the cellular DNA and can exert control when present in vanishingly small quantities may have broad implications in a wide range of infection scenarios. Dysregulation of NID1 in an immunocompetent host is not known to manifest complications during infection; however, in the naive immune system of a developing fetus, disruption of this developmentally critical protein could initiate catastrophic HCMV-induced birth defects.

Study protocol for Video Images about Decisions to Improve Ethical Outcomes with Palliative Care Educators (VIDEO-PCE): a pragmatic stepped wedge cluster randomised trial of older patients admitted to the hospital

INTRODUCTION: Despite the known benefit to patients and families, discussions about goals, values and preferences for medical care in advancing serious illness often do not occur. Many system and clinician factors, such as patient and clinician reticence and shortage of specialty palliative care teams, contribute to this lack of communication. To address this gap, we designed an intervention to promote goals-of-care conversations and palliative care referrals in the hospital setting by using trained palliative care educators and video decision aids. This paper presents the rationale, design and methods for a trial aimed at addressing barriers to goals-of-care conversations for hospitalised adults aged 65 and older and those with Alzheimer's disease and related Dementias, regardless of age. METHODS AND ANALYSIS: The Video Image about Decisions to Improve Ethical Outcomes with Palliative Care Educators is a pragmatic stepped wedge, cluster randomised controlled trial, which aims to improve and extend goals-of-care conversations in the hospital setting with palliative care educators trained in serious illness communication and video decision aids. The primary outcome is the proportion of patients with goals-of-care documentation in the electronic health record. We estimate that over 9000 patients will be included. ETHICS AND DISSEMINATION: The Institutional Review Board (IRB) at Boston Medical Center will serve as the single IRB of record for all regulatory and ethical aspects of this trial. BMC Protocol Number: H-41482. Findings will be presented at national meetings and in publications. This trial is registered at ClinicalTrials.gov. TRIAL REGISTRATION NUMBER: NCT04857060; ClinicalTrials.gov.

The nuclear lamina binds the EBV genome during latency and regulates viral gene expression.

The Epstein Barr virus (EBV) infects almost 95% of the population worldwide. While typically asymptomatic, EBV latent infection is associated with several malignancies of epithelial and lymphoid origin in immunocompromised individuals. In latently infected cells, the EBV genome persists as a chromatinized episome that expresses a limited set of viral genes in different patterns, referred to as latency types, which coincide with varying stages of infection and various malignancies. We have previously demonstrated that latency types correlate with differences in the composition and structure of the EBV episome. Several cellular factors, including the nuclear lamina, regulate chromatin composition and architecture. While the interaction of the viral genome with the nuclear lamina has been studied in the context of EBV lytic reactivation, the role of the nuclear lamina in controlling EBV latency has not been investigated. Here, we report that the nuclear lamina is an essential epigenetic regulator of the EBV episome. We observed that in B cells, EBV infection affects the composition of the nuclear lamina by inducing the expression of lamin A/C, but only in EBV+ cells expressing the Type III latency program. Using ChIP-Seq, we determined that lamin B1 and lamin A/C bind the EBV genome, and their binding correlates with deposition of the histone repressive mark H3K9me2. By RNA-Seq, we observed that knock-out of lamin A/C in B cells alters EBV gene expression. Our data indicate that the interaction between lamins and the EBV episome contributes to the epigenetic control of viral gene expression during latency, suggesting a restrictive function of the nuclear lamina as part of the host response against viral DNA entry into the nucleus.

The three-dimensional structure of Epstein-Barr virus genome varies by latency type and is regulated by PARP1 enzymatic activity.

Epstein-Barr virus (EBV) persists in human B-cells by maintaining its chromatinized episomes within the nucleus. We have previously shown that cellular factor Poly [ADP-ribose] polymerase 1 (PARP1) binds the EBV genome, stabilizes CTCF binding at specific loci, and that PARP1 enzymatic activity correlates with maintaining a transcriptionally active latency program. To better understand PARP1's role in regulating EBV latency, here we functionally characterize the effect of PARP enzymatic inhibition on episomal structure through in situ HiC mapping, generating a complete 3D structure of the EBV genome. We also map intragenomic contact changes after PARP inhibition to global binding of chromatin looping factors CTCF and cohesin across the EBV genome. We find that PARP inhibition leads to fewer total unique intragenomic interactions within the EBV episome, yet new chromatin loops distinct from the untreated episome are also formed. This study also illustrates that PARP inhibition alters gene expression at the regions where chromatin looping is most effected. We observe that PARP1 inhibition does not alter cohesin binding sites but does increase its frequency of binding at those sites. Taken together, these findings demonstrate that PARP has an essential role in regulating global EBV chromatin structure and latent gene expression.

TET2 and DNMT3A Mutations Exert Divergent Effects on DNA Repair and Sensitivity of Leukemia Cells to PARP Inhibitors.

Somatic variants in TET2 and DNMT3A are founding mutations in hematological malignancies that affect the epigenetic regulation of DNA methylation. Mutations in both genes often co-occur with activating mutations in genes encoding oncogenic tyrosine kinases such as FLT3ITD, BCR-ABL1, JAK2V617F , and MPLW515L , or with mutations affecting related signaling pathways such as NRASG12D and CALRdel52 . Here, we show that TET2 and DNMT3A mutations exert divergent roles in regulating DNA repair activities in leukemia cells expressing these oncogenes. Malignant TET2-deficient cells displayed downregulation of BRCA1 and LIG4, resulting in reduced activity of BRCA1/2-mediated homologous recombination (HR) and DNA-PK-mediated non-homologous end-joining (D-NHEJ), respectively. TET2-deficient cells relied on PARP1-mediated alternative NHEJ (Alt-NHEJ) for protection from the toxic effects of spontaneous and drug-induced DNA double-strand breaks. Conversely, DNMT3A-deficient cells favored HR/D-NHEJ owing to downregulation of PARP1 and reduction of Alt-NHEJ. Consequently, malignant TET2-deficient cells were sensitive to PARP inhibitor (PARPi) treatment in vitro and in vivo, whereas DNMT3A-deficient cells were resistant. Disruption of TET2 dioxygenase activity or TET2-Wilms' tumor 1 (WT1)-binding ability was responsible for DNA repair defects and sensitivity to PARPi associated with TET2 deficiency. Moreover, mutation or deletion of WT1 mimicked the effect of TET2 mutation on DSB repair activity and sensitivity to PARPi. Collectively, these findings reveal that TET2 and WT1 mutations may serve as biomarkers of synthetic lethality triggered by PARPi, which should be explored therapeutically. SIGNIFICANCE: TET2 and DNMT3A mutations affect distinct DNA repair mechanisms and govern the differential sensitivities of oncogenic tyrosine kinase-positive malignant hematopoietic cells to PARP inhibitors.

Validation of a Crisis Standards of Care Model for Prioritization of Limited Resources During the Coronavirus Disease 2019 Crisis in an Urban, Safety-Net, Academic Medical Center.

OBJECTIVES: The coronavirus disease 2019 pandemic has overwhelmed healthcare resources even in wealthy nations, necessitating rationing of limited resources without previously established crisis standards of care protocols. In Massachusetts, triage guidelines were designed based on acute illness and chronic life-limiting conditions. In this study, we sought to retrospectively validate this protocol to cohorts of critically ill patients from our hospital. DESIGN: We applied our hospital-adopted guidelines, which defined severe and major chronic conditions as those associated with a greater than 50% likelihood of 1- and 5-year mortality, respectively, to a critically ill patient population. We investigated mortality for the same intervals. SETTING: An urban safety-net hospital ICU. PATIENTS: All adults hospitalized during April of 2015 and April 2019 identified through a clinical database search. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Of 365 admitted patients, 15.89% had one or more defined chronic life-limiting conditions. These patients had higher 1-year (46.55% vs 13.68%; p < 0.01) and 5-year (50.00% vs 17.22%; p < 0.01) mortality rates than those without underlying conditions. Irrespective of classification of disease severity, patients with metastatic cancer, congestive heart failure, end-stage renal disease, and neurodegenerative disease had greater than 50% 1-year mortality, whereas patients with chronic lung disease and cirrhosis had less than 50% 1-year mortality. Observed 1- and 5-year mortality for cirrhosis, heart failure, and metastatic cancer were more variable when subdivided into severe and major categories. CONCLUSIONS: Patients with major and severe chronic medical conditions overall had 46.55% and 50.00% mortality at 1 and 5 years, respectively. However, mortality varied between conditions. Our findings appear to support a crisis standards protocol which focuses on acute illness severity and only considers underlying conditions carrying a greater than 50% predicted likelihood of 1-year mortality. Modifications to the chronic lung disease, congestive heart failure, and cirrhosis criteria should be refined if they are to be included in future models.

EBNA1 inhibitors have potent and selective antitumor activity in xenograft models of Epstein-Barr virus-associated gastric cancer.

BACKGROUND AND AIMS: Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) is the most common EBV-associated cancer and accounts for ~ 10% of all gastric cancers (GC). Epstein-Barr virus nuclear antigen 1 (EBNA1), which is critical for the replication and maintenance of the EBV latent genome, is consistently expressed in all EBVaGC tumors. We previously developed small molecule inhibitors of EBNA1. In this study, we investigated the efficacy and selectivity of an EBNA1 inhibitor in cell-based and animal xenograft models of EBV-positive and EBV-negative gastric carcinoma. METHODS: We tested the potency of an EBNA1 inhibitor, VK-1727, in vitro and in xenograft studies, using EBV-positive (SNU719 and YCCEL1) and EBV-negative (AGS and MKN74) GC cell lines. After treatment, we analyzed cell viability, proliferation, and RNA expression of EBV genes by RT-qPCR. RESULTS: Treatment with VK-1727 selectively inhibits cell cycle progression and proliferation in vitro. In animal studies, treatment with an EBNA1 inhibitor resulted in a significant dose-dependent decrease in tumor growth in EBVaGC xenograft models, but not in EBV-negative GC xenograft studies. Gene expression analysis revealed that short term treatment in cell culture tended towards viral gene activation, while long-term treatment in animal xenografts showed a significant decrease in viral gene expression. CONCLUSIONS: EBNA1 inhibitors are potent and selective inhibitors of cell growth in tissue culture and animal models of EBV-positive GC. Long-term treatment with EBNA1 inhibitors may lead to loss of EBV in mouse xenografts. These results suggest that pharmacological targeting of EBNA1 may be an effective strategy to treat patients with EBVaGC.

Epstein-Barr Virus-Encoded Latent Membrane Protein 1 and B-Cell Growth Transformation Induce Lipogenesis through Fatty Acid Synthase.

Latent membrane protein 1 (LMP1) is the major transforming protein of Epstein-Barr virus (EBV) and is critical for EBV-induced B-cell transformation in vitro Several B-cell malignancies are associated with latent LMP1-positive EBV infection, including Hodgkin's and diffuse large B-cell lymphomas. We have previously reported that promotion of B cell proliferation by LMP1 coincided with an induction of aerobic glycolysis. To further examine LMP1-induced metabolic reprogramming in B cells, we ectopically expressed LMP1 in an EBV-negative Burkitt's lymphoma (BL) cell line preceding a targeted metabolic analysis. This analysis revealed that the most significant LMP1-induced metabolic changes were to fatty acids. Significant changes to fatty acid levels were also found in primary B cells following EBV-mediated B-cell growth transformation. Ectopic expression of LMP1- and EBV-mediated B-cell growth transformation induced fatty acid synthase (FASN) and increased lipid droplet formation. FASN is a crucial lipogenic enzyme responsible for de novo biogenesis of fatty acids in transformed cells. Furthermore, inhibition of lipogenesis caused preferential killing of LMP1-expressing B cells and significantly hindered EBV immortalization of primary B cells. Finally, our investigation also found that USP2a, a ubiquitin-specific protease, is significantly increased in LMP1-positive BL cells and mediates FASN stability. Our findings demonstrate that ectopic expression of LMP1- and EBV-mediated B-cell growth transformation leads to induction of FASN, fatty acids, and lipid droplet formation, possibly pointing to a reliance on lipogenesis. Therefore, the use of lipogenesis inhibitors could be used in the treatment of LMP1+ EBV-associated malignancies by targeting an LMP1-specific dependency on lipogenesis.IMPORTANCE Despite many attempts to develop novel therapies, EBV-specific therapies currently remain largely investigational, and EBV-associated malignancies are often associated with a worse prognosis. Therefore, there is a clear demand for EBV-specific therapies for both prevention and treatment of virus-associated malignancies. Noncancerous cells preferentially obtain fatty acids from dietary sources, whereas cancer cells will often produce fatty acids themselves by de novo lipogenesis, often becoming dependent on the pathway for cell survival and proliferation. LMP1- and EBV-mediated B-cell growth transformation leads to induction of FASN, a key enzyme responsible for the catalysis of endogenous fatty acids. Preferential killing of LMP1-expressing B cells following inhibition of FASN suggests that targeting LMP-induced lipogenesis is an effective strategy in treating LMP1-positive EBV-associated malignancies. Importantly, targeting unique metabolic perturbations induced by EBV could be a way to explicitly target EBV-positive malignancies and distinguish their treatment from EBV-negative counterparts.

TGFßR-SMAD3 Signaling Induces Resistance to PARP Inhibitors in the Bone Marrow Microenvironment.

Synthetic lethality triggered by PARP inhibitor (PARPi) yields promising therapeutic results. Unfortunately, tumor cells acquire PARPi resistance, which is usually associated with the restoration of homologous recombination, loss of PARP1 expression, and/or loss of DNA double-strand break (DSB) end resection regulation. Here, we identify a constitutive mechanism of resistance to PARPi. We report that the bone marrow microenvironment (BMM) facilitates DSB repair activity in leukemia cells to protect them against PARPi-mediated synthetic lethality. This effect depends on the hypoxia-induced overexpression of transforming growth factor beta receptor (TGFßR) kinase on malignant cells, which is activated by bone marrow stromal cells-derived transforming growth factor beta 1 (TGF-ß1). Genetic and/or pharmacological targeting of the TGF-ß1-TGFßR kinase axis results in the restoration of the sensitivity of malignant cells to PARPi in BMM and prolongs the survival of leukemia-bearing mice. Our finding may lead to the therapeutic application of the TGFßR inhibitor in patients receiving PARPis.

Reply to Comment on COVID-19 Deaths in Long Term Care Facilities - A Critical Piece of the Puzzle.

This letter comments on the letter by Tak-kwan Kong.

Poly(ADP-ribosylated) proteins in ß-amyloid peptide-stimulated microglial cells.

Amyloid-treated microglia prime and sustain neuroinflammatory processes in the central nervous system activating different signalling pathways inside the cells. Since a key role for PARP-1 has been demonstrated in inflammation and in neurodegeneration, we investigated PARylated proteins in resting and in ß-amyloid peptide treated BV2 microglial cells. A total of 1158 proteins were identified by mass spectrometry with 117 specifically modified in the amyloid-treated cells. Intervention of PARylation on the proteome of microglia showed to be widespread in different cellular districts and to affect various cellular pathways, highlighting the role of this dynamic post-translational modification in cellular regulation. Ubiquitination is one of the more enriched pathways, encompassing PARylated proteins like NEDD4, an E3 ubiquitine ligase and USP10, a de-ubiquitinase, both associated with intracellular responses induced by ß-amyloid peptide challenge. PARylation of NEDD4 may be involved in the recruiting of this protein to the plasma membrane where it regulates the endocytosis of AMPA receptors, whereas USP10 may be responsible for the increase of p53 levels in amyloid stimulated microglia. Unfolded protein response and Endoplasmic Reticulum Stress pathways, strictly correlated with the Ubiquitination process, also showed enrichment in PARylated proteins. PARylation may thus represent one of the molecular switches responsible for the transition of microglia towards the inflammatory microglia phenotype, a pivotal player in brain diseases including neurodegenerative processes. The establishment of trials with PARP inhibitors to test their efficacy in the containment of neurodegenerative diseases may be envisaged.

Poly(ADP-ribose) polymerase 1 is necessary for coactivating hypoxia-inducible factor-1-dependent gene expression by Epstein-Barr virus latent membrane protein 1.

Latent membrane protein 1 (LMP1) is the major transforming protein of Epstein-Barr virus (EBV) and is critical for EBV-induced B-cell transformation in vitro. Poly(ADP-ribose) polymerase 1 (PARP1) regulates accessibility of chromatin, alters functions of transcriptional activators and repressors, and has been directly implicated in transcriptional activation. Previously we showed that LMP1 activates PARP1 and increases Poly(ADP-ribos)ylation (PARylation) through PARP1. Therefore, to identify targets of LMP1 that are regulated through PARP1, LMP1 was ectopically expressed in an EBV-negative Burkitt's lymphoma cell line. These LMP1-expressing cells were then treated with the PARP inhibitor olaparib and prepared for RNA sequencing. The LMP1/PARP targets identified through this RNA-seq experiment are largely involved in metabolism and signaling. Interestingly, Ingenuity Pathway Analysis of RNA-seq data suggests that hypoxia-inducible factor 1-alpha (HIF-1α) is an LMP1 target mediated through PARP1. PARP1 is acting as a coactivator of HIF-1α-dependent gene expression in B cells, and this co-activation is enhanced by LMP1-mediated activation of PARP1. HIF-1α forms a PARylated complex with PARP1 and both HIF-1α and PARP1 are present at promoter regions of HIF-1α downstream targets, leading to accumulation of positive histone marks at these regions. Complex formation, PARylation and binding of PARP1 and HIF-1α at promoter regions of HIF-1α downstream targets can all be attenuated by PARP1 inhibition, subsequently leading to a buildup of repressive histone marks and loss of positive histone marks. In addition, LMP1 switches cells to a glycolytic 'Warburg' metabolism, preferentially using aerobic glycolysis over mitochondrial respiration. Finally, LMP1+ cells are more sensitive to PARP1 inhibition and, therefore, targeting PARP1 activity may be an effective treatment for LMP1+ EBV-associated malignancies.

PARP1 Stabilizes CTCF Binding and Chromatin Structure To Maintain Epstein-Barr Virus Latency Type.

Epstein Barr virus (EBV) is a potentially oncogenic gammaherpesvirus that establishes a chronic, latent infection in memory B cells. The EBV genome persists in infected host cells as a chromatinized episome and is subject to chromatin-mediated regulation. Binding of the host insulator protein CTCF to the EBV genome has an established role in maintaining viral latency type. CTCF is posttranslationally modified by the host enzyme PARP1. PARP1, or poly(ADP-ribose) polymerase 1, catalyzes the transfer of a poly(ADP-ribose) (PAR) moiety from NAD+ onto acceptor proteins, including itself, histone proteins, and CTCF. PARylation of CTCF by PARP1 can affect CTCF's insulator activity, DNA binding capacity, and ability to form chromatin loops. Both PARP1 and CTCF have been implicated in the regulation of EBV latency and lytic reactivation. Thus, we predicted that pharmacological inhibition with PARP1 inhibitors would affect EBV latency type through a chromatin-specific mechanism. Here, we show that PARP1 and CTCF colocalize at specific sites throughout the EBV genome and provide evidence to suggest that PARP1 acts to stabilize CTCF binding and maintain the open chromatin landscape at the active Cp promoter during type III latency. Further, PARP1 activity is important in maintaining latency type-specific viral gene expression. The data presented here provide a rationale for the use of PARP inhibitors in the treatment of EBV-associated cancers exhibiting type III latency and ultimately could contribute to an EBV-specific treatment strategy for AIDS-related or posttransplant lymphomas.IMPORTANCE EBV is a human gammaherpesvirus that infects more than 95% of individuals worldwide. Upon infection, EBV circularizes as an episome and establishes a chronic, latent infection in B cells. In doing so, the virus utilizes host cell machinery to regulate and maintain the viral genome. In otherwise healthy individuals, EBV infection is typically nonpathological; however, latent infection is potentially oncogenic and is responsible for 1% of human cancers. During latent infection, EBV expresses specific sets of proteins according to the given latency type, each of which is associated with specific types of cancers. For example, type III latency, in which the virus expresses its full repertoire of latent proteins, is characteristic of AIDS-associated and posttransplant lymphomas associated with EBV infection. Understanding how viral latency type is regulated at the chromatin level may reveal potential targets for EBV-specific pharmacological intervention in EBV-associated cancers.