Lytic Cycle I : Linda van Dyk and Mei-Ru Chen
Oral Talk #8
Continuous DNA Replication Is Required for Late Gene
Transcription and Maintenance of Replication Compartments in
Gammaherpesviruses
Dajiang Li1, Wenmin Fu1, Sankar Swaminathan1,2
1Division of Infectious Diseases, Department of Medicine, University of Utah School of Medicine, 2George E. Wahlen Department of Veterans Affairs Medical Center
Late gene transcription in herpesviruses is dependent on viral DNA replication in cis but the mechanistic basis for this linkage remains unknown. DNA replication results in demethylated DNA, topological changes, removal of proteins and recruitment of proteins to promoters. One or more of these effects of DNA replication may facilitate late gene transcription. Using 5-azacytidine to promote demethylation of DNA, we demonstrate that late gene transcription cannot be rescued by DNA demethylation. Late gene transcription precedes significant increases in DNA copy number, indicating that increased template numbers also do not contribute to the linkage between replication and late gene transcription. By using serial, timed blockade of DNA replication and measurement of late gene mRNA accumulation, we demonstrate that late gene transcription requires ongoing DNA replication. Consistent with these findings, blocking DNA replication led to dissolution of DNA replication complexes which also contain RNA polymerase II and BGLF4, an EBV protein required for transcription of several late genes. These data indicate that ongoing DNA replication maintains integrity of a replication-transcription complex which is required for recruitment and retention of factors necessary for late gene transcription.
Continuous DNA Replication Is Required for Late Gene
Transcription and Maintenance of Replication Compartments in
Gammaherpesviruses
Dajiang Li1, Wenmin Fu1, Sankar Swaminathan1,2
1Division of Infectious Diseases, Department of Medicine, University of Utah School of Medicine, 2George E. Wahlen Department of Veterans Affairs Medical Center
Late gene transcription in herpesviruses is dependent on viral DNA replication in cis but the mechanistic basis for this linkage remains unknown. DNA replication results in demethylated DNA, topological changes, removal of proteins and recruitment of proteins to promoters. One or more of these effects of DNA replication may facilitate late gene transcription. Using 5-azacytidine to promote demethylation of DNA, we demonstrate that late gene transcription cannot be rescued by DNA demethylation. Late gene transcription precedes significant increases in DNA copy number, indicating that increased template numbers also do not contribute to the linkage between replication and late gene transcription. By using serial, timed blockade of DNA replication and measurement of late gene mRNA accumulation, we demonstrate that late gene transcription requires ongoing DNA replication. Consistent with these findings, blocking DNA replication led to dissolution of DNA replication complexes which also contain RNA polymerase II and BGLF4, an EBV protein required for transcription of several late genes. These data indicate that ongoing DNA replication maintains integrity of a replication-transcription complex which is required for recruitment and retention of factors necessary for late gene transcription.
Oral Talk #9
Intracellular Iron Chelation by a Novel Compound, C7, Induces
Lytic Cycle of Epstein-Barr Virus (EBV) in EBV-Positive
Epithelial Malignancies
P.T. Stephanie Yiu1, Kwai Fung Hui1, 4, Miriam Choi1, Richard Kao2, Dan Yang3, Alan K.S.Chiang1, 4
1Department of Paediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam, 2Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 3Department of Chemistry, The University of Hong Kong
Pharmaceutical reactivation of Epstein-Barr virus (EBV) lytic cycle represents a potential EBV-targeted therapeutic strategy against EBV-associated malignancies including gastric carcinoma (GC) and nasopharyngeal carcinoma (NPC). A novel lytic inducing compound, C7, which contains a transitional metal binding domain, had been identified from a high- throughput screening. This study aims to investigate the ability of C7 and different iron chelators to induce EBV lytic cycle in EBV-positive GC and NPC cells. Testing of 6 structural analogues of C7 revealed that only those analogues which displayed high affinity towards iron could induce EBV lytic cycle in the EBV-positive GC and NPC cells. We further showed that Dp44mT, an established iron chelator structurally similar to C7, and the clinically available iron chelators such as deferoxamine, deferiprone and deferasirox, which are structurally distinct from C7, could induce EBV lytic cycle in the GC and NPC cells.Intracellular iron chelation was demonstrated using calcein fluorescent probe and the chelation had been further shown to be essential for lytic induction activity of C7 and the clinical iron chelators as pre-complexing these chemicals to iron prior to treatment abolished their reactivation of EBV lytic cycle. Transcriptome profile analysis revealed the enrichment of the hypoxia pathway as the hallmark gene set upon C7 treatment in GC cells, further supporting the correlation between intracellular iron chelation and EBV lytic reactivation. C7 and the iron chelators could also sensitize EBV-positive GC cells to killing by an anti-viral agent, ganciclovir. In summary, intracellular iron chelation by a novel compound, C7, could potently induce lytic cycle of EBV in EBV-positive epithelial malignancies.
Oral Talk #10
Application of the Air Liquid Interface Culture Method to Study
EBV Pathogenesis in Polarized Airway Epithelia
Kathy Shair1,2, Elizabeth Caves1, Sarah Cook1, Nara Lee2, Donna Stoltz3, Simon Watkins3
1Cancer Virology Program, UPMC Hillman Cancer Center, 2Department of Microbiology &
Molecular Genetics, University of Pittsburgh, 3Department of Cell Biology, University of Pittsburgh
Epstein-Barr virus (EBV) latent infection is associated with the epithelial cancer nasopharyngeal carcinoma (NPC). EBV infection of oral epithelial cells primarily results in a lytic infection, which likely contributes to the transmission of EBV from saliva, but the mechanisms governing EBV infection outcome in the mucosal nasopharyngeal epithelium are largely undetermined. The EBV LMP1 latent protein is a pleiotropic signaling molecule that induces oncogenic properties in multiple cell types, however the contribution of LMP1 to EBV pathogenesis has not been elucidated. A better understanding could be gained from a 3D epithelial culture model to study EBV pathogenesis in polarized airway epithelium. The recent application of organotypic rafts has demonstrated great promise for the use of polarized cultures in the study of EBV permissive replication in keratinocytes. Here, we present evidence demonstrating that the air liquid interface (ALI) culture method on transwell membranes is fully permissive with production of progeny virus. Global induction of the lytic cascade, viral genome amplification and production of encapsidated progeny virus can be examined by analysis of RNA, protein, DNA and infectious virus harvested from the ALI method. The effect of LMP1 expression on permissive replication was revealed using the ALI method, indicating that LMP1 contributes to efficient EBV permissive replication. The ALI method can uniquely preserve the diversity of cell types representing nasal epithelium from primary cultures. We propose that the polarized airway culture model can be widely adopted to study EBV-infection outcomes in established epithelial cell lines and discuss the application of the ALI method to study de-novo infection in primary nasal epithelial cells.
EBV Pathogenesis in Polarized Airway Epithelia
Kathy Shair1,2, Elizabeth Caves1, Sarah Cook1, Nara Lee2, Donna Stoltz3, Simon Watkins3
1Cancer Virology Program, UPMC Hillman Cancer Center, 2Department of Microbiology &
Molecular Genetics, University of Pittsburgh, 3Department of Cell Biology, University of Pittsburgh
Epstein-Barr virus (EBV) latent infection is associated with the epithelial cancer nasopharyngeal carcinoma (NPC). EBV infection of oral epithelial cells primarily results in a lytic infection, which likely contributes to the transmission of EBV from saliva, but the mechanisms governing EBV infection outcome in the mucosal nasopharyngeal epithelium are largely undetermined. The EBV LMP1 latent protein is a pleiotropic signaling molecule that induces oncogenic properties in multiple cell types, however the contribution of LMP1 to EBV pathogenesis has not been elucidated. A better understanding could be gained from a 3D epithelial culture model to study EBV pathogenesis in polarized airway epithelium. The recent application of organotypic rafts has demonstrated great promise for the use of polarized cultures in the study of EBV permissive replication in keratinocytes. Here, we present evidence demonstrating that the air liquid interface (ALI) culture method on transwell membranes is fully permissive with production of progeny virus. Global induction of the lytic cascade, viral genome amplification and production of encapsidated progeny virus can be examined by analysis of RNA, protein, DNA and infectious virus harvested from the ALI method. The effect of LMP1 expression on permissive replication was revealed using the ALI method, indicating that LMP1 contributes to efficient EBV permissive replication. The ALI method can uniquely preserve the diversity of cell types representing nasal epithelium from primary cultures. We propose that the polarized airway culture model can be widely adopted to study EBV-infection outcomes in established epithelial cell lines and discuss the application of the ALI method to study de-novo infection in primary nasal epithelial cells.
Oral Talk #11
NLRX1 Negatively Modulates Type I IFN to Facilitate KSHV
Reactivation from Latency
Zhe Ma1, Sharon Hopcraft1, Fan Yang1, Alex Petrucelli1, Haitao Guo1, Jenny Ting1,2, Dirk Dittmer1,2, Blossom Damania1,2
1 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, 2Department of Microbiology and Immunology, University of North Carolina at Chapel Hill
Kaposi’s sarcoma-associated herpesvirus (KSHV) is a herpesvirus that is linked to Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD). KSHV establishes persistent latent infection in the human host. KSHV undergoes periods of spontaneous reactivation where it can enter the lytic replication phase of its lifecycle. During KSHV reactivation, host innate immune responses are activated to restrict viral replication. Here, we report that NLRX1, a negative regulator of the type I interferon response, is important for optimal KSHV reactivation from latency.Depletion of NLRX1 in either iSLK.219 or BCBL-1 cells significantly suppressed global viral transcription levels compared to the control group. Concomitantly, fewer viral particles were present in either cells or supernatant from NLRX1 depleted cells. Further analysis revealed that upon NLRX1 depletion, higher IFNβ transcription levels were observed, which was also associated with a transcriptional upregulation of JAK/STAT pathway related genes in both cell lines. To investigate whether IFNβ contributes to NLRX1’s role in KSHV reactivation, we treated control and NLRX1 depleted cells with a TBK1 inhibitor (BX795) or TBK1 siRNA to block IFNβ production. Upon BX795 or TBK1 siRNA treatment, NLRX1 depletion exhibited less inhibitory effects on reactivation and infectious virion production, suggesting that NLRX1 facilitates KSHV lytic replication by negatively regulating IFNβ responses. Our data suggests that NLRX1 plays a positive role in KSHV lytic replication by suppressing the IFNβ response during the process of KSHV reactivation, which might serve as a potential target for restricting KSHV replication and transmission.
NLRX1 Negatively Modulates Type I IFN to Facilitate KSHV
Reactivation from Latency
Zhe Ma1, Sharon Hopcraft1, Fan Yang1, Alex Petrucelli1, Haitao Guo1, Jenny Ting1,2, Dirk Dittmer1,2, Blossom Damania1,2
1 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, 2Department of Microbiology and Immunology, University of North Carolina at Chapel Hill
Kaposi’s sarcoma-associated herpesvirus (KSHV) is a herpesvirus that is linked to Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD). KSHV establishes persistent latent infection in the human host. KSHV undergoes periods of spontaneous reactivation where it can enter the lytic replication phase of its lifecycle. During KSHV reactivation, host innate immune responses are activated to restrict viral replication. Here, we report that NLRX1, a negative regulator of the type I interferon response, is important for optimal KSHV reactivation from latency.Depletion of NLRX1 in either iSLK.219 or BCBL-1 cells significantly suppressed global viral transcription levels compared to the control group. Concomitantly, fewer viral particles were present in either cells or supernatant from NLRX1 depleted cells. Further analysis revealed that upon NLRX1 depletion, higher IFNβ transcription levels were observed, which was also associated with a transcriptional upregulation of JAK/STAT pathway related genes in both cell lines. To investigate whether IFNβ contributes to NLRX1’s role in KSHV reactivation, we treated control and NLRX1 depleted cells with a TBK1 inhibitor (BX795) or TBK1 siRNA to block IFNβ production. Upon BX795 or TBK1 siRNA treatment, NLRX1 depletion exhibited less inhibitory effects on reactivation and infectious virion production, suggesting that NLRX1 facilitates KSHV lytic replication by negatively regulating IFNβ responses. Our data suggests that NLRX1 plays a positive role in KSHV lytic replication by suppressing the IFNβ response during the process of KSHV reactivation, which might serve as a potential target for restricting KSHV replication and transmission.
Oral Talk #12
BGLF2 Increases Infectivity of Epstein-Barr virus by Activating
AP-1 Upon de Novo Infection
Takayuki Murata1,2, Natsuno Konishi2, Yohei Narita2, Yoshitaka Sato2, Hiroshi Kimura2
1Fujita Health University School of Medicine, 2Nagoya University Graduate School of Medicine
Herpesviruses encode genes that can modify cell functions, such as transcription and cell death, thereby facilitating viral growth and survival in infected cells. Using a reporter screening system, we revealed the involvement of several EBV gene products in such processes. Of these, the BGLF2 activated AP-1signaling pathway through phosphorylation of p38 and JNK. Knockout of the BGLF2 gene did not affect viral gene expression and viral genome DNA replication, but resulted in mild decrease of progeny virion production into medium and marked loss in infectivity of the produced virion. Interestingly, expression of a binding partner, BKRF4, repressed the activation of AP-1 by BGLF2. Taken together, we speculate that, in late phase of lytic cycle when elevated AP-1 activation is perhaps not needed anymore, BKRF4 sequesters and inhibits BGLF2. BGLF2 and BKRF4 proteins are incorporated into tegument, and BGLF2 is released in newly infected cells to activate AP-1 pathway and increases transcription of viral genes, especially during the pre-latent phase.
BGLF2 Increases Infectivity of Epstein-Barr virus by Activating
AP-1 Upon de Novo Infection
Takayuki Murata1,2, Natsuno Konishi2, Yohei Narita2, Yoshitaka Sato2, Hiroshi Kimura2
1Fujita Health University School of Medicine, 2Nagoya University Graduate School of Medicine
Herpesviruses encode genes that can modify cell functions, such as transcription and cell death, thereby facilitating viral growth and survival in infected cells. Using a reporter screening system, we revealed the involvement of several EBV gene products in such processes. Of these, the BGLF2 activated AP-1signaling pathway through phosphorylation of p38 and JNK. Knockout of the BGLF2 gene did not affect viral gene expression and viral genome DNA replication, but resulted in mild decrease of progeny virion production into medium and marked loss in infectivity of the produced virion. Interestingly, expression of a binding partner, BKRF4, repressed the activation of AP-1 by BGLF2. Taken together, we speculate that, in late phase of lytic cycle when elevated AP-1 activation is perhaps not needed anymore, BKRF4 sequesters and inhibits BGLF2. BGLF2 and BKRF4 proteins are incorporated into tegument, and BGLF2 is released in newly infected cells to activate AP-1 pathway and increases transcription of viral genes, especially during the pre-latent phase.
Oral Talk #13
The M81 Epstein-Barr Virus Associated RNA 2 Enhances CXCL8 Production and Spontaneous Virus Lytic Replication in
Primary B Lymphocytes
Zhe Li1,2,3, Ming-Han Tsai1,3,4, Anatoliy Shumilov1,3, Rémy Poirey1,3, Henri-Jacques Delecluse1,2,3
1German Cancer Research Center (DKFZ), Unit F100, 2Faculty of Biosciences, Heidelberg University, 3The Institut national de la santé et de la recherche médicale (INSERM) Unit U1074, 4Institute of Microbiology and Immunology, National Yang-Ming University
The Epstein-Barr virus (EBV) infects the majority of the population. The EBV M81 strain efficiently infects and transforms primary B cells, but it also induces potent virus lytic replication in a minority of these cells. We found that the number of cells in which lytic replication takes place is increased both in vitro and in vivo by the non-coding RNA EBER2, but not by its homolog in the B95-8 genome. Both RNAs display a limited number of polymorphisms, some of which influence their half-life and expression levels. M81 EBER2 modified the expression of a large number of cellular genes including CXCL8. This chemokine was able to compensate the absence of EBER2, suggesting that it represents the main target of this non-coding RNA. We found that the exosomal fraction of B cells infected with wild type M81 released in the extracellular milieu carry the EBER molecules, are able to increase CXCL8 and BZLF1 production and thus partly complement the phenotype of B cells infected with a virus that lacks the EBER RNAs. The effect of EBER2 on EBV lytic replication required a functional TLR7, a sensor of viral single-stranded RNA (ssRNA). Therefore, we propose a model in which EBERs are vehicled into the exosomal fraction of infected B cells to initiate lytic replication in a paracrine manner through CXCL8 secretion induced by TLR7 stimulation. These results indicate that EBERs NPC-derived virus variant contribute to lytic replication in B cells and activate production of a chemokine involved in carcinogenesis.
Session 2B: Therapeutics & Vaccines
Session Chairs: Robert Yarchoan & Mu-sheng Zeng
The M81 Epstein-Barr Virus Associated RNA 2 Enhances CXCL8 Production and Spontaneous Virus Lytic Replication in
Primary B Lymphocytes
Zhe Li1,2,3, Ming-Han Tsai1,3,4, Anatoliy Shumilov1,3, Rémy Poirey1,3, Henri-Jacques Delecluse1,2,3
1German Cancer Research Center (DKFZ), Unit F100, 2Faculty of Biosciences, Heidelberg University, 3The Institut national de la santé et de la recherche médicale (INSERM) Unit U1074, 4Institute of Microbiology and Immunology, National Yang-Ming University
The Epstein-Barr virus (EBV) infects the majority of the population. The EBV M81 strain efficiently infects and transforms primary B cells, but it also induces potent virus lytic replication in a minority of these cells. We found that the number of cells in which lytic replication takes place is increased both in vitro and in vivo by the non-coding RNA EBER2, but not by its homolog in the B95-8 genome. Both RNAs display a limited number of polymorphisms, some of which influence their half-life and expression levels. M81 EBER2 modified the expression of a large number of cellular genes including CXCL8. This chemokine was able to compensate the absence of EBER2, suggesting that it represents the main target of this non-coding RNA. We found that the exosomal fraction of B cells infected with wild type M81 released in the extracellular milieu carry the EBER molecules, are able to increase CXCL8 and BZLF1 production and thus partly complement the phenotype of B cells infected with a virus that lacks the EBER RNAs. The effect of EBER2 on EBV lytic replication required a functional TLR7, a sensor of viral single-stranded RNA (ssRNA). Therefore, we propose a model in which EBERs are vehicled into the exosomal fraction of infected B cells to initiate lytic replication in a paracrine manner through CXCL8 secretion induced by TLR7 stimulation. These results indicate that EBERs NPC-derived virus variant contribute to lytic replication in B cells and activate production of a chemokine involved in carcinogenesis.
Session 2B: Therapeutics & Vaccines
Session Chairs: Robert Yarchoan & Mu-sheng Zeng
Oral Talk #14
Toward the Clinical Development of an EBNA1 Inhibitor for the
Treatment of EBV-Associated Nasopharyngeal Carcinoma
Troy E. Messick1, Samantha S. Soldan1, Garry R. Smith2, Julianna S. Deakyne1, Kimberly A. Malecka1, Lois Tolvinski1, Pierre Busson3, Allen B. Reitz2, Paul M. Lieberman1
1The Wistar Institute, 2Fox Chase Chemical Diversity Center, 3Institut Gustave Roussy
New, more effective and less toxic treatments are needed for patients with EBV-associated nasopharyngeal carcinoma (NPC). Tumorigenesis and continued tumor growth of NPC is dependent on the continuous expression of Epstein-Barr Nuclear Antigen 1 (EBNA1), a multifunctional dimeric protein critical for viral replication, genome maintenance and viral gene expression. The aim of this program is to advance the development of a New Chemical Entity (NCE) to eliminate latent EBV as a treatment for NPC. We have used structure-based drug design and medicinal chemistry methods to identify and develop a small molecule clinical candidate that selectively inhibits the DNA-binding activity of EBNA1. The clinical candidate inhibits EBNA1 function with nanomolar potency in biochemical assays and low micromolecular activity in several cell-based assays. We demonstrate that our clinical candidate provides protection in 4 different xenograft models of EBV-driven tumor growth, including NPC patient-derived xenografts. Furthermore, RNA analysis experiments confirm in vivo target engagement by the elimination of EBV in treated tumor tissue. The clinical candidate is selective, showing no activity in an EBV- negative xenograft experiments. The clinical candidate has met industry-accepted criteria for drug suitability, safety and toxicology including physicochemical properties, metabolic stability, selectivity in broad-based screens and bioavailability, and lack in vivo liabilities, including genotoxicity and 28-day toxicity studies. IND-enabling studies including safety pharmacology and toxicology and GMP manufacturing have been completed. We chose patients with advanced NPC as patient population because (1) NPC is ~100% EBV- positive; (2) systemic therapies for metastatic or locally recurrent NPC presently offer response rates of 40-60% with median survivals of only 20 months, with a substantial toxicity profile; and (3) EBV plasma levels can be used as a biomarker to measure early response to treatment. These data establish proof-of-concept for targeting EBNA1—a program we anticipate beginning first-in-human studies in H2 2018.
Treatment of EBV-Associated Nasopharyngeal Carcinoma
Troy E. Messick1, Samantha S. Soldan1, Garry R. Smith2, Julianna S. Deakyne1, Kimberly A. Malecka1, Lois Tolvinski1, Pierre Busson3, Allen B. Reitz2, Paul M. Lieberman1
1The Wistar Institute, 2Fox Chase Chemical Diversity Center, 3Institut Gustave Roussy
New, more effective and less toxic treatments are needed for patients with EBV-associated nasopharyngeal carcinoma (NPC). Tumorigenesis and continued tumor growth of NPC is dependent on the continuous expression of Epstein-Barr Nuclear Antigen 1 (EBNA1), a multifunctional dimeric protein critical for viral replication, genome maintenance and viral gene expression. The aim of this program is to advance the development of a New Chemical Entity (NCE) to eliminate latent EBV as a treatment for NPC. We have used structure-based drug design and medicinal chemistry methods to identify and develop a small molecule clinical candidate that selectively inhibits the DNA-binding activity of EBNA1. The clinical candidate inhibits EBNA1 function with nanomolar potency in biochemical assays and low micromolecular activity in several cell-based assays. We demonstrate that our clinical candidate provides protection in 4 different xenograft models of EBV-driven tumor growth, including NPC patient-derived xenografts. Furthermore, RNA analysis experiments confirm in vivo target engagement by the elimination of EBV in treated tumor tissue. The clinical candidate is selective, showing no activity in an EBV- negative xenograft experiments. The clinical candidate has met industry-accepted criteria for drug suitability, safety and toxicology including physicochemical properties, metabolic stability, selectivity in broad-based screens and bioavailability, and lack in vivo liabilities, including genotoxicity and 28-day toxicity studies. IND-enabling studies including safety pharmacology and toxicology and GMP manufacturing have been completed. We chose patients with advanced NPC as patient population because (1) NPC is ~100% EBV- positive; (2) systemic therapies for metastatic or locally recurrent NPC presently offer response rates of 40-60% with median survivals of only 20 months, with a substantial toxicity profile; and (3) EBV plasma levels can be used as a biomarker to measure early response to treatment. These data establish proof-of-concept for targeting EBNA1—a program we anticipate beginning first-in-human studies in H2 2018.
Oral Talk #15
The Unique Structure of the LMP1 – TRAF6 Complex is Critical
for Viral Cell Transformation and Serves as Target for
Inhibitory Molecules
Fabian Giehler1,2, Michael Ostertag3, Thomas Sommermann4, Daniel Weidl5, Kai R. Sterz1, Brigitte Biesinger5, Klaus Rajewsky4, Grzegorz Popowicz3, Johannes Kirchmair6, Arnd Kieser1,2
1Helmholtz Center Munich – German Research Center For Environmental Health, 2German Center for Infection Research (DZIF) – Partner Site Munich, 3Helmholtz Center Munich – German Research Center for Environmental Health, Institute of Structural Biology, 4Max-Delbruck-Center for Molecular Medicine, Immune Regulation and Cancer, 5Friedrich-Alexander University, Institute of Clinical and Molecular Virology, 6University of Hamburg, Center for Bioinformatics, Research Group for Applied Cheminformatics and Molecular Design
The latent membrane protein 1 (LMP1) is the primary oncogene of EBV. LMP1 activates NF-κB and JNK by engaging TNF-receptor-associated factor 6 (TRAF6) as the essential signaling mediator of the C-terminal activation region 2 (CTAR2). However, until today it is unclear how TRAF6 interacts with LMP1. Here, we aimed at elucidating the molecular mechanism of LMP1–TRAF6 interaction and its relevance for LMP1 signaling and function. Using recombinant TRAF6 and LMP1 proteins in two-component in vitro binding assays including a newly established alphascreen-based protein-protein interaction assay, we show that TRAF6 in fact binds directly to a cryptic TRAF motif within CTAR2. This motif matches the NF-κB and JNK activation motifs of CTAR2 and is distantly related to the TRAF6 binding site of CD40. However, structural modelling of the LMP1–TRAF6 complex, which is supported by 2D-NMR data and mutational analysis, shows that significant differences between LMP1 and CD40 binding to TRAF6 exist. TRAF6 point mutants that are unable to bind to LMP1 in vitro also fail to form cellular complexes with LMP1. Furthermore, these mutants are unable to rescue NF-κB signaling by LMP1 in TRAF6-/- cells, confirming the relevance of direct LMP1-TRAF6 interaction for CTAR2 function also in vivo. Notably, the CRISPR/Cas9-mediated knockout of TRAF6 in LMP1-driven B- lymphoma cells derived from LMP1-transgenic mice interferes with survival of the lymphoma cells. Altogether these data suggest that the direct LMP1–TRAF6 interaction is a potential novel target structure for inhibitory small molecules. As proof-of-concept we show that an inhibitory peptide blocks LMP1 interaction with TRAF6 in our alphascreen- based high throughput screening assay and interferes with survival of EBV-transformed human B-cells. Taken together, we have identified the molecular basis of LMP1 interaction with its essential signaling mediator TRAF6 and validated this direct interaction as a new target for inhibitors.
for Viral Cell Transformation and Serves as Target for
Inhibitory Molecules
Fabian Giehler1,2, Michael Ostertag3, Thomas Sommermann4, Daniel Weidl5, Kai R. Sterz1, Brigitte Biesinger5, Klaus Rajewsky4, Grzegorz Popowicz3, Johannes Kirchmair6, Arnd Kieser1,2
1Helmholtz Center Munich – German Research Center For Environmental Health, 2German Center for Infection Research (DZIF) – Partner Site Munich, 3Helmholtz Center Munich – German Research Center for Environmental Health, Institute of Structural Biology, 4Max-Delbruck-Center for Molecular Medicine, Immune Regulation and Cancer, 5Friedrich-Alexander University, Institute of Clinical and Molecular Virology, 6University of Hamburg, Center for Bioinformatics, Research Group for Applied Cheminformatics and Molecular Design
The latent membrane protein 1 (LMP1) is the primary oncogene of EBV. LMP1 activates NF-κB and JNK by engaging TNF-receptor-associated factor 6 (TRAF6) as the essential signaling mediator of the C-terminal activation region 2 (CTAR2). However, until today it is unclear how TRAF6 interacts with LMP1. Here, we aimed at elucidating the molecular mechanism of LMP1–TRAF6 interaction and its relevance for LMP1 signaling and function. Using recombinant TRAF6 and LMP1 proteins in two-component in vitro binding assays including a newly established alphascreen-based protein-protein interaction assay, we show that TRAF6 in fact binds directly to a cryptic TRAF motif within CTAR2. This motif matches the NF-κB and JNK activation motifs of CTAR2 and is distantly related to the TRAF6 binding site of CD40. However, structural modelling of the LMP1–TRAF6 complex, which is supported by 2D-NMR data and mutational analysis, shows that significant differences between LMP1 and CD40 binding to TRAF6 exist. TRAF6 point mutants that are unable to bind to LMP1 in vitro also fail to form cellular complexes with LMP1. Furthermore, these mutants are unable to rescue NF-κB signaling by LMP1 in TRAF6-/- cells, confirming the relevance of direct LMP1-TRAF6 interaction for CTAR2 function also in vivo. Notably, the CRISPR/Cas9-mediated knockout of TRAF6 in LMP1-driven B- lymphoma cells derived from LMP1-transgenic mice interferes with survival of the lymphoma cells. Altogether these data suggest that the direct LMP1–TRAF6 interaction is a potential novel target structure for inhibitory small molecules. As proof-of-concept we show that an inhibitory peptide blocks LMP1 interaction with TRAF6 in our alphascreen- based high throughput screening assay and interferes with survival of EBV-transformed human B-cells. Taken together, we have identified the molecular basis of LMP1 interaction with its essential signaling mediator TRAF6 and validated this direct interaction as a new target for inhibitors.
Oral Talk #16
CRISPR Resistance Screens Reveal Genetic Mediators of
Immunomodulatory Drugs in Primary Effusion Lymphoma
Ajinkya Patil1, Mark Manzano1, Eva Gottwein1
1Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University
The immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide have recently been proposed as therapeutic options for the treatment of the KSHV-associated malignancy primary effusion lymphoma (PEL). IMiDs bind to the CRBN substrate adaptor of the CUL4- CRBN E3 ubiquitin ligase complex to redirect its activities to novel targets, including IKZF1, IKZF3, CK1α and ZFP91. We have recently shown that IMiDs also induce the indirect downregulation of the oncogene IRF4 in PEL cells, independently of the degradation of known IMiD neosubstrates. Here we report an unbiased investigation of the mechanisms of IMiDs in PEL cells using CRISPR/Cas9-based screens for resistance of BC-3 cells to the IMiDs lenalidomide, pomalidomide and CC-122. Our screens consistently identified CRBN as the most dominant hit, underscoring IMiD specificity and the central role of CUL4-CRBN E3 ligase in IMiD toxicity. Furthermore, we identified several novel regulators of the CUL4- CRBN E3 ligase complex that mediate IMiD activity. Inactivation of the E2 ubiquitin conjugating enzyme UBE2G1 in BC-3 cells conferred significant resistance against IMiDs and rescued IMiD-induced degradation of IMiD neosubstrates and more importantly, downregulation of IRF4. Our data thus suggest that UBE2G1 is likely the E2 ligase for CUL4-CRBN. Similarly, individual inactivation of the subunits of the CUL4 complex, CUL4A or CUL4B, and of the Nedd8-specific protease SENP8 conferred significant resistance to lenalidomide but not pomalidomide. In summary, our CRISPR resistance screens identified several genetic mediators of IMiDs in PEL cells in vitro. Our approach facilitated the identification of novel regulators of CRL4CBRN, including one of its E2 ubiquitin ligases. Genes whose inactivation confers IMiD resistance could be developed as biomarkers to predict the response to IMiDs in the treatment of PEL and other malignancies.
Immunomodulatory Drugs in Primary Effusion Lymphoma
Ajinkya Patil1, Mark Manzano1, Eva Gottwein1
1Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University
The immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide have recently been proposed as therapeutic options for the treatment of the KSHV-associated malignancy primary effusion lymphoma (PEL). IMiDs bind to the CRBN substrate adaptor of the CUL4- CRBN E3 ubiquitin ligase complex to redirect its activities to novel targets, including IKZF1, IKZF3, CK1α and ZFP91. We have recently shown that IMiDs also induce the indirect downregulation of the oncogene IRF4 in PEL cells, independently of the degradation of known IMiD neosubstrates. Here we report an unbiased investigation of the mechanisms of IMiDs in PEL cells using CRISPR/Cas9-based screens for resistance of BC-3 cells to the IMiDs lenalidomide, pomalidomide and CC-122. Our screens consistently identified CRBN as the most dominant hit, underscoring IMiD specificity and the central role of CUL4-CRBN E3 ligase in IMiD toxicity. Furthermore, we identified several novel regulators of the CUL4- CRBN E3 ligase complex that mediate IMiD activity. Inactivation of the E2 ubiquitin conjugating enzyme UBE2G1 in BC-3 cells conferred significant resistance against IMiDs and rescued IMiD-induced degradation of IMiD neosubstrates and more importantly, downregulation of IRF4. Our data thus suggest that UBE2G1 is likely the E2 ligase for CUL4-CRBN. Similarly, individual inactivation of the subunits of the CUL4 complex, CUL4A or CUL4B, and of the Nedd8-specific protease SENP8 conferred significant resistance to lenalidomide but not pomalidomide. In summary, our CRISPR resistance screens identified several genetic mediators of IMiDs in PEL cells in vitro. Our approach facilitated the identification of novel regulators of CRL4CBRN, including one of its E2 ubiquitin ligases. Genes whose inactivation confers IMiD resistance could be developed as biomarkers to predict the response to IMiDs in the treatment of PEL and other malignancies.
Oral Talk #17
Dual Role in Fighting Viral Malignancies: Next Generation
Epigenetic Modifier for Viral Gene Activation and Anti-Tumor
Immune Response
Robert A Baiocchi1, Robert C. McRae2, Preston Warren2, Christopher C. Oakes1, Christoph Weigel1, Sarah Schlotter1, Marshelle Smith Warren2
1Division of Hematology, Department of Internal Medicine, The Ohio State University, 2Viracta Therapeutics
The concept of using histone deacetylase inhibitors (HDI) to induce Epstein-Barr virus (EBV) lytic genes in conjunction with antiviral prodrugs to be activated by viral kinases is well established. However, off-target effects and immune suppression were observed with first-generation HDIs.
Tractinostat is a selective HDI optimized to induce expression of epigenetically silenced EBV genes. In vitro studies using an EBV positive non-Hodgkin’s lymphoma cell line, NK-92, there was no significant single agent activity with tractinostat or ganciclovir alone, however, complete elimination of cells was observed when these agents were combined. Selective anti-tumor toxicity was associated with induction of the EBV lytic gene BZLF1 and the kinases BXLF1 (vTK) and BGLF4 (homologue to UL97 CMV kinase). This “kick and kill” combination therapy is currently being investigated in a Ph1b/2 clinical trial for EBV positive lymphomas (NCT03397706). A previous proof of concept study using arginine butyrate, a first generation HDI greater than 1000-fold less potent for target gene induction, demonstrated a 67% ORR in 21 patients of various lymphoma etiologies receiving ≤ 21 days of treatment. The tractinostat study will combine 2 orally available compounds, tractinostat and valganciclovir, and is planning to enroll 30 – 45 patients with EBV+ lymphoproliferative diseases (LPD). Eligibility will include patients with LPDs associated with iatrogenic immune suppression due to solid organ or hematopoietic cell transplant, acquired immunodeficiency, including HIV positive, and other LPDs not associated with immunodeficiency. HDAC isoforms responsible for EBV latency are shared with those that regulate immune function and T cell exhaustion. Tractinostat has demonstrated unique immune modulator qualities, driving a shift from a Th2 to Th1 type responses, enhancing the anti-viral and anti-tumor immune response in ex vivo primary cancer patient cells and in vivo murine models. Synergistically, this approach may release viral and tumor neoepitopes targetable by the host immune system.
Epigenetic Modifier for Viral Gene Activation and Anti-Tumor
Immune Response
Robert A Baiocchi1, Robert C. McRae2, Preston Warren2, Christopher C. Oakes1, Christoph Weigel1, Sarah Schlotter1, Marshelle Smith Warren2
1Division of Hematology, Department of Internal Medicine, The Ohio State University, 2Viracta Therapeutics
The concept of using histone deacetylase inhibitors (HDI) to induce Epstein-Barr virus (EBV) lytic genes in conjunction with antiviral prodrugs to be activated by viral kinases is well established. However, off-target effects and immune suppression were observed with first-generation HDIs.
Tractinostat is a selective HDI optimized to induce expression of epigenetically silenced EBV genes. In vitro studies using an EBV positive non-Hodgkin’s lymphoma cell line, NK-92, there was no significant single agent activity with tractinostat or ganciclovir alone, however, complete elimination of cells was observed when these agents were combined. Selective anti-tumor toxicity was associated with induction of the EBV lytic gene BZLF1 and the kinases BXLF1 (vTK) and BGLF4 (homologue to UL97 CMV kinase). This “kick and kill” combination therapy is currently being investigated in a Ph1b/2 clinical trial for EBV positive lymphomas (NCT03397706). A previous proof of concept study using arginine butyrate, a first generation HDI greater than 1000-fold less potent for target gene induction, demonstrated a 67% ORR in 21 patients of various lymphoma etiologies receiving ≤ 21 days of treatment. The tractinostat study will combine 2 orally available compounds, tractinostat and valganciclovir, and is planning to enroll 30 – 45 patients with EBV+ lymphoproliferative diseases (LPD). Eligibility will include patients with LPDs associated with iatrogenic immune suppression due to solid organ or hematopoietic cell transplant, acquired immunodeficiency, including HIV positive, and other LPDs not associated with immunodeficiency. HDAC isoforms responsible for EBV latency are shared with those that regulate immune function and T cell exhaustion. Tractinostat has demonstrated unique immune modulator qualities, driving a shift from a Th2 to Th1 type responses, enhancing the anti-viral and anti-tumor immune response in ex vivo primary cancer patient cells and in vivo murine models. Synergistically, this approach may release viral and tumor neoepitopes targetable by the host immune system.
Oral Talk #18
Modulating IL-7 Cytokine Receptor Signaling to Enhance the
Persistence and Anti-Tumor Efficacy of Epstein-Barr Virus
(EBV) Specific T-Cells in EBV Positive Malignancies
Sandhya Sharma1, Thomas Shum1, Bilal Omer1,2, Mai Huynh1,2, Cliona M. Rooney1,2 1Baylor College of Medicine, 2Texas Childrens Hospital
Up to ~40 % of lymphomas carry the Epstein-Barr virus (EBV) genome involving the expression of EBNA-1, LMP-1, LMP-2 and BARF-1 (type 2 latency viral antigens [T2Ags]). Our lab has used T-cells specific for T2Ags (T2EBVSTs) to target EBV positive lymphoma with promising results. However, lack of cytokines to support T-cell growth in vivo, and suppressive tumor microenvironment (TME) are major challenges to the persistence and efficacy of T2EBVSTs. IL-7 has both growth promoting and anti-apoptotic effects on T-cells and may be exploited for the benefit of T2EBVSTs. Hence, we hypothesize that T2EBVSTs expressing constitutively active IL-7 receptor (C7R) to enable cytokine-independent proliferation will have increased persistence and potency in EBV+ malignancies.
T2EBVSTs expressing C7R (65%±25% transduction, n=6), were generated with stable specificity for T2Ags (5064±2988 vs. 4683±2134 SFCs per 105 T2EBVSTs, ELISpot assay; C7R-T2EBVSTs vs. unmodified T2EBVSTs). C7R-T2EBVSTs demonstrated enhanced STAT5-signaling (STAT5 MFI of 778±66 vs. 139±9) and enhanced in-vitro persistence in cytokine starved culture conditions (35±2 days vs. 16±3 days for unmodified T2EBVSTs). To evaluate the anti-tumor effects of C7R-T2EBVSTs, I established subcutaneous EBV transformed B-cells (EBV-LCLs) murine xenograft models and (tumor volume-130 mm3), infused C7R-T2EBVSTs or unmodified EBVSTs. Mice receiving C7R-T2EBVSTs exhibited tumor clearance by day 60, while the tumor volume of mice receiving unmodified T2EBVSTs was 240 ± 62 mm3; p<0 .000="" 27="" 39="" 602="" 67="" also="" c7r-t2ebvsts="" control="" day="" days="" demonstrated="" did="" enhanced="" for="" mice="" mm3="" n="5/group)." not="" on="" persistence="" receive="" sacrificed="" span="" t2ebvsts="" that="" umor="" unmodified="" volume="" vs.="" were="">
The demonstration of increased persistence and enhanced anti-tumor efficacy of T2EBVSTs expressing C7R in our EBV positive tumor model provides a novel and innovative platform for translating our findings to future clinical trials. In future experiments, we will elucidate the mechanisms underlying the protective effects of C7R in the presence of suppressive TME.
Persistence and Anti-Tumor Efficacy of Epstein-Barr Virus
(EBV) Specific T-Cells in EBV Positive Malignancies
Sandhya Sharma1, Thomas Shum1, Bilal Omer1,2, Mai Huynh1,2, Cliona M. Rooney1,2 1Baylor College of Medicine, 2Texas Childrens Hospital
Up to ~40 % of lymphomas carry the Epstein-Barr virus (EBV) genome involving the expression of EBNA-1, LMP-1, LMP-2 and BARF-1 (type 2 latency viral antigens [T2Ags]). Our lab has used T-cells specific for T2Ags (T2EBVSTs) to target EBV positive lymphoma with promising results. However, lack of cytokines to support T-cell growth in vivo, and suppressive tumor microenvironment (TME) are major challenges to the persistence and efficacy of T2EBVSTs. IL-7 has both growth promoting and anti-apoptotic effects on T-cells and may be exploited for the benefit of T2EBVSTs. Hence, we hypothesize that T2EBVSTs expressing constitutively active IL-7 receptor (C7R) to enable cytokine-independent proliferation will have increased persistence and potency in EBV+ malignancies.
T2EBVSTs expressing C7R (65%±25% transduction, n=6), were generated with stable specificity for T2Ags (5064±2988 vs. 4683±2134 SFCs per 105 T2EBVSTs, ELISpot assay; C7R-T2EBVSTs vs. unmodified T2EBVSTs). C7R-T2EBVSTs demonstrated enhanced STAT5-signaling (STAT5 MFI of 778±66 vs. 139±9) and enhanced in-vitro persistence in cytokine starved culture conditions (35±2 days vs. 16±3 days for unmodified T2EBVSTs). To evaluate the anti-tumor effects of C7R-T2EBVSTs, I established subcutaneous EBV transformed B-cells (EBV-LCLs) murine xenograft models and (tumor volume-130 mm3), infused C7R-T2EBVSTs or unmodified EBVSTs. Mice receiving C7R-T2EBVSTs exhibited tumor clearance by day 60, while the tumor volume of mice receiving unmodified T2EBVSTs was 240 ± 62 mm3; p<0 .000="" 27="" 39="" 602="" 67="" also="" c7r-t2ebvsts="" control="" day="" days="" demonstrated="" did="" enhanced="" for="" mice="" mm3="" n="5/group)." not="" on="" persistence="" receive="" sacrificed="" span="" t2ebvsts="" that="" umor="" unmodified="" volume="" vs.="" were="">
The demonstration of increased persistence and enhanced anti-tumor efficacy of T2EBVSTs expressing C7R in our EBV positive tumor model provides a novel and innovative platform for translating our findings to future clinical trials. In future experiments, we will elucidate the mechanisms underlying the protective effects of C7R in the presence of suppressive TME.
Oral Talk #19
Spironolactone Blocks EBV Production by Degrading XPB
Protein, A Cellular Transcription Factor that Acts as a Cofactor
for EBV SM Function
Dinesh Verma1, Trenton M Church1, Sankar Swaminathan1,2
1Division of Infectious Diseases, University of Utah School of Medicine, 2Department of Pathology, University of Utah School of Medicine
Epstein Barr virus (EBV) is a human gammaherpesvirus associated with several lymphoid and epithelial tumors. Most clinically available drugs active against herpesviruses target viral DNA polymerase and have limitations of toxicity and development of resistance. EBV SM is a nuclear EBV protein, essential for lytic viral replication and production of infectious virions. SM enhances expression of several EBV lytic genes in addition to regulating host cell gene expression. We have shown that SM effects on EBV target are gene-specific and that SM is preferentially required for expression of a subset of EBV late lytic genes. EBV SM homologous genes have been described in all human herpesviruses including herpes simplex virus, human cytomegalovirus, and Kaposi’s sarcoma associated herpesvirus (KSHV). Therefore, investigating the mechanisms of action of SM, and targeting SM protein and its homologs in other herpesviruses may prove to be a useful strategy for developing molecular therapies for herpesvirus infections with minimal off-target effects on host cells.
We used a cell based, high-throughput screening assay and demonstrated that spironolactone (SPR), a clinically used heart failure drug, blocks EBV virion production by inhibiting EBV SM function. While SM binds to and stabilizes some target RNAs, and has long been thought to act post-transcriptionally only, we provide here new evidence that SM also acts to facilitate EBV gene transcription. Our data suggest that SM acts at the transcription initiation or elongation step and that SPR specifically inhibits this function by targeting a cellular transcription factor XPB, that acts as a cofactor for EBV lytic cycle gene expression. We have found that SPR acts to destabilize a cellular transcription factor, XPB, that appears to be uniquely involved in EBV gene transcription. SPR is therefore hypothesized to act through effects on host cell transcription factors that are necessary for SM function.
Spironolactone Blocks EBV Production by Degrading XPB
Protein, A Cellular Transcription Factor that Acts as a Cofactor
for EBV SM Function
Dinesh Verma1, Trenton M Church1, Sankar Swaminathan1,2
1Division of Infectious Diseases, University of Utah School of Medicine, 2Department of Pathology, University of Utah School of Medicine
Epstein Barr virus (EBV) is a human gammaherpesvirus associated with several lymphoid and epithelial tumors. Most clinically available drugs active against herpesviruses target viral DNA polymerase and have limitations of toxicity and development of resistance. EBV SM is a nuclear EBV protein, essential for lytic viral replication and production of infectious virions. SM enhances expression of several EBV lytic genes in addition to regulating host cell gene expression. We have shown that SM effects on EBV target are gene-specific and that SM is preferentially required for expression of a subset of EBV late lytic genes. EBV SM homologous genes have been described in all human herpesviruses including herpes simplex virus, human cytomegalovirus, and Kaposi’s sarcoma associated herpesvirus (KSHV). Therefore, investigating the mechanisms of action of SM, and targeting SM protein and its homologs in other herpesviruses may prove to be a useful strategy for developing molecular therapies for herpesvirus infections with minimal off-target effects on host cells.
We used a cell based, high-throughput screening assay and demonstrated that spironolactone (SPR), a clinically used heart failure drug, blocks EBV virion production by inhibiting EBV SM function. While SM binds to and stabilizes some target RNAs, and has long been thought to act post-transcriptionally only, we provide here new evidence that SM also acts to facilitate EBV gene transcription. Our data suggest that SM acts at the transcription initiation or elongation step and that SPR specifically inhibits this function by targeting a cellular transcription factor XPB, that acts as a cofactor for EBV lytic cycle gene expression. We have found that SPR acts to destabilize a cellular transcription factor, XPB, that appears to be uniquely involved in EBV gene transcription. SPR is therefore hypothesized to act through effects on host cell transcription factors that are necessary for SM function.