2018 Madison Meeting
HENLE LECTURE
Paul M. Lieberman, Ph.D. The Wistar Institute Philadelphia, PA
Paul Lieberman obtained a Ph.D. at Johns Hopkins University, School of Medicine studying Epstein-Barr Virus reactivation in the laboratory of Dr. Gary and Diane Hayward. He completed a post-doctoral fellowship with Dr. Arnie Berk at UCLA investigating basic mechanisms of transcriptional activation by viral factors. He joined the Roche Institute of Molecular Biology as an Assistant Member and then moved to the Wistar Institute in Philadelphia in 1995. His research focuses on the molecular biology of Epstein-Barr Virus (EBV) and Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) latency and persistence, with particular interests in epigenetic mechanisms and chromosome biology. His work combines biochemical, proteomic, and functional genomic methods to investigate regulation of viral latency, and how this contributes to viral carcinogenesis. The Lieberman group has leveraged this basic research to develop novel small molecule inhibitors and modulators of EBV and KSHV latency to treat viral-associated disease. Lieberman is presently the Hilary Koprowski Endowed Chair and Program Leader in Gene Expression and Regulation and the Director of the Center for Chemical Biology and Translational Medicine at the Wistar Institute.
Arch Pharm Res. 2017 Aug;40(8):894-905. doi: 10.1007/s12272-017-0939-5. Epub 2017 Aug 4.
DNA hypermethylation induced by Epstein-Barr virus in the development of Epstein-Barr virus-associated gastric carcinoma.
Abstract
Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) is a recently recognized disease entity defined by the presence of EBV in gastric carcinoma cells. EBV infection causes major epigenetic alterations in the EBV genome and its cellular host genome, suggesting that EBV acts as a direct epigenetic driver for EBVaGC.
One of the major epigenetic events in the viral and cellular genomes to control transcription is DNA hypo- or hyper-methylation. Particularly, local and global hypermethylation have been reported in EBVaGC. It is therefore important to understand the molecular mechanisms of DNA hypermethylation during EBVaGC carcinogenesis. To understand the functional roles of DNA methylation and suggest therapeutic target candidates for EBVaGC, we reviewed recent literature reporting DNA hypermethylation in EBVaGC.
We summarized the identified candidate genes that are markedly hypermethylated in EBVaGC, which can potentially be targets for chemotherapies with demethylating agents. (pmid:28779374)
J Virol. 2017 Aug 9. pii: JVI.00804-17. doi: 10.1128/JVI.00804-17. [Epub ahead of print]
Coordinate Regulation of TET2 and EBNA2 Control DNA Methylation State of Latent Epstein-Barr Virus.
Abstract
Epstein-Barr Virus (EBV) latency and its associated carcinogenesis are regulated by dynamic changes in DNA methylation of both virus and host genomes. We show here that the Ten-Eleven Translocation 2 (TET2) gene, implicated in hydroxymethylation and active DNA demethylation, is a key regulator of EBV latency type DNA methylation patterning. EBV latency types are defined by DNA methylation patterns that restrict expression of viral latency genes. We show that TET2 mRNA and protein expression correlate with the highly demethylated EBV type III latency program permissive for expression of EBNA2, EBNA3s, and LMP transcripts. We show that shRNA depletion of TET2 results in a decrease in latency gene expression, but can also trigger a switch to lytic gene expression. TET2 depletion results in the loss of hydroxymethylated cytosine, and corresponding increase in cytosine methylation at key regulatory regions on the viral and host genomes. This also corresponded to a loss of RBP-jκ binding, and decreased histone H3K4 trimethylation at these sites. Furthermore, we show that the TET2 gene, itself, is regulated similar to the EBV genome. ChIP-Seq revealed that TET2 gene contains EBNA2-dependent RBP-jκ and EBF1 binding sites, and is subject to DNA methylation associated transcriptional silencing similar to EBV latency type III genomes. Finally, we provide evidence that TET2 colocalizes with EBNA2-EBF1-RBP-jκ binding sites, and can interact with EBNA2 by co-immunoprecipitation. Taken together, these findings indicate that TET2 gene transcripts are regulated similarly to EBV type III latency genes, and that TET2 protein is a cofactor of EBNA2 and co-regulator of the EBV type III latency program and DNA methylation state.
IMPORTANCE Epstein-Barr Virus(EBV) latency and carcinogenesis involves the selective epigenetic modification of viral and cellular genes.
Here, we show that TET2, a cellular tumor suppressor involved in active DNA demethylation, plays a central role in regulating DNA methylation state during EBV latency. TET2 is coordinately regulated and functionally interacts with the viral oncogene EBNA2.
TET2 and EBNA2 function cooperatively to demethylate genes important for EBV-driven B cells growth transformation. (
PMID: 28794029)
PLoS Pathog. 2017 Aug 30;13(8):e1006596. doi: 10.1371/journal.ppat.1006596. eCollection 2017 Aug.
Deregulation of KSHV latency conformation by ER-stress and caspase-dependent RAD21-cleavage.
Abstract
Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV) is a human gammaherpesvirus recognized as the principal causative agent of KS and primary effusion lymphoma (PEL). KSHV establishes persistent latent infection in B lymphocytes where viral gene expression is restricted, in part, by a cohesin-dependent chromosome conformation.
Here, we show that endoplasmic reticulum (ER) stress induces a rapid, caspase-dependent cleavage of cohesin subunit RAD21. E
R stress-induced cleavage of RAD21 correlated with a rapid and strong viral lytic transcriptional activation. This effect was observed in several KSHV positive PEL cells, but not in other B-cells or non-B-cell models of KSHV latency. The cleaved-RAD21 does not dissociate from viral genomes, nor disassemble from other components of the cohesin complex.
However, RAD21 cleavage correlated with the disruption of the latency genome conformation as revealed by chromosome conformation capture (3C). Ectopic expression of C-terminal RAD21 cleaved form could partially induce KSHV lytic genes transcription in BCBLI cells, suggesting that ER-stress induced RAD21 cleavage was sufficient to induce KSHV reactivation from latency in PEL cells. Taken together our results reveal a novel aspect for control and maintenance of KSHV genome latency conformation mediated by stress-induced RAD21 cleavage.
Our studies also suggest that RAD21 cleavage may be a general regulatory mechanism for rapid alteration of cellular chromosome conformation and cohesin-dependent transcription regulation. (
PubMed: 28854249)
PLoS Pathog. 2018 Jan 19;14(1):e1006844. doi: 10.1371/journal.ppat.1006844. eCollection 2018 Jan.
RNA-Seq of Kaposi’s sarcoma reveals alterations in glucose and lipid metabolism.
Tso FY1,
Kossenkov AV2,
Lidenge SJ1,3,4,
Ngalamika O5,
Ngowi JR3,
Mwaiselage J3,4,
Wickramasinghe J2,
Kwon EH1,
West JT1,
Lieberman PM2,
Wood C1.
Abstract
Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi’s sarcoma (KS). It is endemic in a number of sub-Saharan African countries with infection rate of >50%. The high prevalence of HIV-1 coupled with late presentation of advanced cancer staging make KS the leading cancer in the region with poor prognosis and high mortality. Disease markers and cellular functions associated with KS tumorigenesis remain ill-defined.
Several studies have attempted to investigate changes of the gene profile with in vitro infection of monoculture models, which are not likely to reflect the cellular complexity of the in vivo lesion environment. Our approach is to characterize and compare the gene expression profile in KS lesions versus non-cancer tissues from the same individual. Such comparisons could identify pathways critical for KS formation and maintenance. T
his is the first study that utilized high throughput RNA-seq to characterize the viral and cellular transcriptome in tumor and non-cancer biopsies of African epidemic KS patients. These patients were treated anti-retroviral therapy with undetectable HIV-1 plasma viral load. We found remarkable variability in the viral transcriptome among these patients, with viral latency and immune modulation genes most abundantly expressed.
The presence of KSHV also significantly affected the cellular transcriptome profile. Specifically, genes involved in lipid and glucose metabolism disorder pathways were substantially affected. Moreover, infiltration of immune cells into the tumor did not prevent KS formation, suggesting some functional deficits of these cells. Lastly, we found only minimal overlaps between our in vivo cellular transcriptome dataset with those from in vitro studies, reflecting the limitation of in vitro models in representing tumor lesions. These findings could lead to the identification of diagnostic and therapeutic markers for KS, and will provide bases for further mechanistic studies on the functions of both viral and cellular genes that are involved. (
PubMed: 29352292)
2016 GRC NPC
2016 EBV Meeting Session 4: Latent EBV proteins
Functions of EBNA1 in Chromatin Regulation
Paul Lieberman,The Wistar Institute, Philadelphia, USA”
—————–
PLoS Pathogens -Research Matters (2016/07) <全文連結>
How do viruses cause cancer? As a naïve graduate student in the years before kits and personal computers, I found myself fascinated by viral “immortalization,” the process by which a virus induces a cell to live forever. The immortalizing virus of interest was named Epstein–Barr Virus (EBV) after its co-discoverers and was isolated from tumors commonly found in African children. EBV was also known to be a major cause of infectious mononucleosis, commonly referred to as the “kissing disease,” and a potential agent in chronic fatigue syndromes. For these reasons, I suspect, EBV was slow to get the respect it deserved. We now know that EBV is found in diverse tumors and is responsible for almost ~200,000 cancer cases each year. It was also tempting to speculate over a beer whether viral immortalization could reveal fresh insights into human mortality and life span. On more sober occasions, the question of how a virus could cause cancer seemed a big enough challenge.
Over the years, my interest in viral immortalization morphed into an investigation of viral gene regulation and latency. How does EBV establish long-term latent infection and express only a few essential viral genes necessary to keep the host cell dividing and alive? To get at these questions, we needed to combine many different methods ranging from functional genomics to structural biology, requiring expertise in diverse disciplines far beyond my capabilities. Building a team of collaborators with the appropriate expertise and identifying the most appropriate technologies is essential. Selecting the right tools and team to answer the bigger questions is not always straightforward, but establishing multi-disciplinary collaborations can open many otherwise closed doors.
Among the most important and challenging steps in a research career is identifying a significant long-term research problem. This sounds easy, but it has many snags. Highly ambitious long-term goals, like curing EBV cancers, may not be achievable without more mundane short-term groundwork. And short-term goals, like the need to get the next paper published or grant funded, can sometimes distract from the long-term plan. Working on tangential problems is necessary to overcome obstacles and can lead to new and unanticipated discoveries. But keeping sight of the long-term goal can be a great strength for a research program. If all goes well, the research focus will be aligned with reviewers and funding agencies. Fortunately for EBV research, foundations like Cancer Research UK have recently recognized the eradication of EBV-associated cancers as one of the decade’s grand challenges. So how best to respond to this grand challenge?
From my perspective, studying the basic mechanisms of EBV latency is the best path to identify viral-specific targets for therapeutic intervention. At least half of my group is committed to developing small molecules to either inhibit EBV latency or stimulate viral reactivation, two possible strategies to treat latent viruses in cancer cells. Viral reactivation from latency is attractive because it is likely to trigger the host immune system to eliminate EBV-infected cancer cells. Inhibiting latency should lead to the loss of viral DNA from cancer cells that depend on EBV for growth and survival. In principle, inhibitors of EBV latency or activators of viral lytic antigens should be valuable therapies to treat EBV-associated cancer and related disease.
Of course these more ambitious projects are risky and require vast sums of funding. The pharmaceutical industry and venture capitalists have no tolerance for financing the early stages of long-term, high-risk projects, so public funding of academic investigators will be the only possible path forward. Although there are many academics who consider translational research a corruption of academic freedom, the urgency of real-world unmet medical needs becomes very apparent if one only takes a look at the economic and human cost of disease. It will be essential for academic investigators to fulfill these obligations to society. Ironically, an editorial opposing the funding of basic academic research was recently written by Matt Ridley in The Wall Street Journal arguing that public sponsorship of basic research blocks private investment and derails innovation. In fact, the only chance for innovative solutions to long-term challenges will be through public funding of basic academic research. Encouraging scientists to focus on unmet medical needs as well as conceptual advances in basic science should be a high priority for public and private investment in a healthy future.
——————
MAY 20, 2015
(l to r): Paul Lieberman, Ph.D.; Troy Messick, Ph.D.
PHILADELPHIA—(May 20, 2015)— The
Wistar Institute and London-based global charity
Wellcome Trust announce that they have signed a follow-on funding agreement in support of ongoing research and development of a new class of drugs useful for treating cancers associated with Epstein-Barr virus (EBV). The Seeding Drug Discovery Award of up to $5.6 million, over the next three years, will support ongoing translational research in the laboratory of
Paul M. Lieberman, Ph.D., Hilary Koprowski, M.D., Endowed Professor, Professor and Program Leader, Gene Expression and Regulation Program, and Director, Center for Chemical Biology and Translational Medicine at Wistar.
If successful, this novel therapeutic could be the first to treat EBV-related cancers by attacking the virus as it remains dormant in a patient’s cells. It is estimated that EBV causes nearly 200,000 cases of cancer worldwide each year, including Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, gastric carcinoma, and certain head and neck cancers.
The project was initially funded in 2011 as a three-year, multi-stage effort with ongoing financial support based on the achievement of defined research milestones. Since then, Wistar investigators and their drug development partners, the Fox Chase Chemical Diversity Center in Doylestown, Pennsylvania, and DavosPharma in Upper Saddle River, New Jersey, have developed lead candidate therapeutic molecules demonstrating proof of concept in animal models. With this renewed grant money, they will continue optimizing these leads for safety, potency and efficacy with an eye toward assembling an Investigational New Drug package for the Food and Drug Administration, which would permit testing of the therapeutic candidate in a Phase 1 clinical trial.
“On behalf of The Wistar Institute, I would like to thank the Wellcome Trust for its continuing support and significant contributions made to advancing our EBV targeted cancer therapeutic program,” said Dario Altieri, M.D., Wistar’s president and CEO, and Cancer Center director. “This effort reflects translational research at its finest by combining a visionary charitable foundation, a promising line of research, and a solid plan for transforming basic science into practical medicine.”
Approximately 90 percent of adults in the U.S. have antibodies indicating a current or past infection with EBV. EBV, the first human DNA tumor virus discovered, can linger in the human body for decades before causing infected cells to become cancerous. With limited therapeutic options for these cancers, long-term prognosis is poor.
EBNA1, which stands for Epstein-Barr virus Nuclear Antigen-1, is the only viral protein expressed consistently in all EBV-related cancers and is essential for the virus to reproduce, making it a prime target for therapeutic intervention.
“EBNA1 acts as a ‘safe house’ that keeps the viral DNA protected and active in tumor cells,” said Lieberman. “Inhibiting EBNA1, therefore, should eliminate the viral DNA and prevent the growth of EBV-associated cancer.” Lieberman is encouraged that his lab’s work may also have value against other chronic diseases associated with EBV, such as multiple sclerosis and infectious mononucleosis.
To develop an anti-EBV drug, researchers began a complex screening process to find a small molecule that could chemically bind to EBNA1 and inhibit its ability to function. They began with a library of small “fragment” compounds. Using atomic resolution structures of fragments with EBNA1, they were eventually able to “build out” the fragments into leading candidate molecules that selectively inhibit the growth of EBV-infected cells.
With funds and intellectual contributions provided by the Wellcome Trust, Wistar researchers, together with their drug development expert network, will further optimize their small molecule inhibitors, with the aim of developing at least one chemical compound into a viable drug candidate. This drug candidate could then be used in clinical trials designed to determine its safety and effectiveness in humans.
“This is an investment in drug discovery, enabling a small team of experts to do the type of translational research typically seen in large drug companies,” said Troy Messick, Ph.D., Senior Staff Scientist in the Lieberman laboratory and co-leader on the project. “If successful with the translational research funded by the Wellcome Trust, the program will be in a position to attract a commercial partner to undertake further clinical development.”
About the Wistar Institute:
The Wistar Institute is an international leader in biomedical research with special expertise in cancer research and vaccine development. Founded in 1892 as the first independent nonprofit biomedical research institute in the country, Wistar has long held the prestigious Cancer Center designation from the National Cancer Institute. The Institute works actively to ensure that research advances move from the laboratory to the clinic as quickly as possible. Wistar Science Saves Lives. www.wistar.org.About the Wellcome Trust:
The Wellcome Trust is a global charitable foundation dedicated to improving health. The Trust provides more than $1.1 billion a year to support bright minds in science, the humanities and the social sciences, as well as education, public engagement and the application of research to medicine. www.wellcome.ac.uk. For more information on Seeding Drug Discovery funding, please visit the Wellcome Trust website: http://j.mp/SeedingDrugDiscovery
Troy Messick (The Wistar Institute, USA)
“Development of Small Molecule Inhibitors of EBNA1 for the Treatment of Nasopharyngeal Carcinoma”” (https://goo.gl/5njCTt) (5.6 millions USD!!)
From Ming-Han
Model screening: by xenograft.
Work well for MutiLCLs; nearly 90% tumor disappear
Work fine for NPC tumor; around 60-70% tumor size smaller.
Studying the EBV Xenograft tumor (smaller one after treatment) comparing to the non-treated: all EBV mRNA level 10x less.
Now: preclinical trials (high oral formula), to screen toxicity
@@Eurofins Cerep Safety Screen: Company can do all the screening for you for preclinical trials!!
@@Safty-> GMP-> preclinical-> clinical-> 2018 April