Session Chairs: Thomas Schulz (replaced by a lady) and Micah Luftig
Oral Talk #78
Increased Association between EBV EBNA2 from Type 2
Strains and the Transcriptional Repressor BS69 Restricts B
Cell Growth
Rajesh Ponnusamy1, Ritika Khatri1, Paolo Correia2, Erika Mancini1, Paul J. Farrell2, Michelle J. West1
1School of Life Sciences, University of Sussex,, 2Section of Virology, Imperial College
Natural variation separates Epstein-Barr virus (EBV) into type 1 and type 2 strains. Type 1 EBV transforms B lymphocytes in vitro more efficiently than type 2 and sequence differences in the EBV transcription factor EBNA2 determine this phenotype. The reduced transforming activity of type 2 EBNA2 correlates with reduced binding and activation of the viral oncogene LMP1 and some cellular genes. Transcriptional activation by type 1 EBNA2 can be suppressed by interaction with the dimeric tumour suppressor protein BS69 (ZMYND11). Two MYND domains of BS69 bind to two adjacent PXLXP motifs in type 1 EBNA2. We identified a third PXLXP BS69 interaction motif in type 2 EBNA2, implicating enhanced BS69 binding in restricting gene activation and transformation. Using isothermal titration calorimetry we demonstrated that a peptide encompassing the third motif from type 2 EBNA2 bound to BS69 and that a region of type 2 EBNA2 containing all three PXLXP motifs bound to BS69 with a stoichiometry indicative of the binding of an additional BS69 dimer. Full-length type 2 EBNA2 also bound more efficiently to BS69 in pull-down assays. Size exclusion chromatography with multi-angle light scattering confirmed the binding of an additional BS69 dimer to type 2 EBNA2. It also demonstrated that both type 1 and type 2 EBNA2 can form higher-order complexes with BS69 involving two molecules of EBNA2, consistent with the dimeric state of EBNA2 in vivo. Small-angle X-ray scattering generated low-resolution solution structures consistent with the binding of an additional BS69 dimer to type 2 EBNA2. Importantly, mutation of the third BS69 binding motif in type 2 EBNA2 improved the ability of type 2 EBNA2 to maintain B cell growth. Taken together, our data indicate that increased association with BS69 may restrict gene activation and contribute to the reduced transformation efficiency of type 2 EBV.
Strains and the Transcriptional Repressor BS69 Restricts B
Cell Growth
Rajesh Ponnusamy1, Ritika Khatri1, Paolo Correia2, Erika Mancini1, Paul J. Farrell2, Michelle J. West1
1School of Life Sciences, University of Sussex,, 2Section of Virology, Imperial College
Natural variation separates Epstein-Barr virus (EBV) into type 1 and type 2 strains. Type 1 EBV transforms B lymphocytes in vitro more efficiently than type 2 and sequence differences in the EBV transcription factor EBNA2 determine this phenotype. The reduced transforming activity of type 2 EBNA2 correlates with reduced binding and activation of the viral oncogene LMP1 and some cellular genes. Transcriptional activation by type 1 EBNA2 can be suppressed by interaction with the dimeric tumour suppressor protein BS69 (ZMYND11). Two MYND domains of BS69 bind to two adjacent PXLXP motifs in type 1 EBNA2. We identified a third PXLXP BS69 interaction motif in type 2 EBNA2, implicating enhanced BS69 binding in restricting gene activation and transformation. Using isothermal titration calorimetry we demonstrated that a peptide encompassing the third motif from type 2 EBNA2 bound to BS69 and that a region of type 2 EBNA2 containing all three PXLXP motifs bound to BS69 with a stoichiometry indicative of the binding of an additional BS69 dimer. Full-length type 2 EBNA2 also bound more efficiently to BS69 in pull-down assays. Size exclusion chromatography with multi-angle light scattering confirmed the binding of an additional BS69 dimer to type 2 EBNA2. It also demonstrated that both type 1 and type 2 EBNA2 can form higher-order complexes with BS69 involving two molecules of EBNA2, consistent with the dimeric state of EBNA2 in vivo. Small-angle X-ray scattering generated low-resolution solution structures consistent with the binding of an additional BS69 dimer to type 2 EBNA2. Importantly, mutation of the third BS69 binding motif in type 2 EBNA2 improved the ability of type 2 EBNA2 to maintain B cell growth. Taken together, our data indicate that increased association with BS69 may restrict gene activation and contribute to the reduced transformation efficiency of type 2 EBV.
EBNA2
- Doesn’t bind DNA directly
- Interacts with cellular
Type 2 EBNA2 is a weaker activator if certain genes
- LMP1 was activated better by type 1 EBNA2 than type 2
A single amino acid in EBNA2 affects LCL growth maintenance (S442D)
BS69 (ZMYND11)
- Multi-domain chromatin associated repoiresor protien that suppresses transcription elongation , regulates pre0mRNA processing has typo
- Contains PHD
- BS69 MYND domain builds type 1 EBNA2
- Type 1 EBNA2 Harter et al PLoS pathogens 2016
- Does the D to in motif 2 of TADs make difference in binding to BS69 CC-MYNDS (it is likely to influence conformation change in the protein but does not affect BS69 association)
- SAXS small angle x-ray scattering
- Type 2 EBNA2 increases association with BS69 via additional motif (dimers of EBNA2 associates with trimers of BS69)
- Loss of the third BS69 binding motif in Type 2 EBNA2 improves LCL growth maintenance function
Oral Talk #79
The Oncogenic Kaposi’s Sarcoma-Associated Herpesvirus
Encodes a Mimic of the Tumor Suppressor miR-16
Kylee Morrison1, Mark Manzano1, Kevin Chung1, Matthew J. Schipma2, Eva Gottwein1
1Department of Microbiology-Immunology, Northwestern University, Feinberg School of Medicine, 2Department of Biochemistry and Molecular Genetics, Northwestern University, Feinberg School of Medicine
Kaposi’s sarcoma-associated herpesvirus (KSHV) causes several cancers, including Kaposi’s sarcoma and primary effusion B cell lymphoma. KSHV expresses numerous viral microRNAs (miRNAs) during the latent phase of infection, including viral mimics of miR- 155, miR-142-3p and miR-23. Recently, we noticed an extended, but offset, sequence similarity between the KSHV miRNA miR-K6-5p and the tumor suppressor cellular miR-16. Here, we have characterized the relationship between miR-K6-5p and miR-16. RNA- sequencing and extensive validation experiments show that miR-K6-5p and miR-16 both use canonical seed matching for target regulation. At the target level, miR-K6-5p was able to regulate most miR-16 targets, in some instances at altered efficiencies compared to miR-16. Additionally, miR-K6-5p regulates a small number of targets that are not shared with miR-16. At the functional level, miR-K6-5p shared tumor suppressive functions of miR- 16, including the induction of cell cycle arrest. Altogether, our data suggest that this oncogenic herpesvirus encodes a functional mimic of miR-16. Our ongoing experiments address the hypothesis that miR-K6-5p functions to balance consequences of viral oncogene expression. While many oncogenic herpesviruses encode gene products that antagonize tumor suppressors, this is – to our knowledge – the first example of an oncogenic virus that encodes a homolog or mimic of a bona fide tumor suppressor.
Encodes a Mimic of the Tumor Suppressor miR-16
Kylee Morrison1, Mark Manzano1, Kevin Chung1, Matthew J. Schipma2, Eva Gottwein1
1Department of Microbiology-Immunology, Northwestern University, Feinberg School of Medicine, 2Department of Biochemistry and Molecular Genetics, Northwestern University, Feinberg School of Medicine
Kaposi’s sarcoma-associated herpesvirus (KSHV) causes several cancers, including Kaposi’s sarcoma and primary effusion B cell lymphoma. KSHV expresses numerous viral microRNAs (miRNAs) during the latent phase of infection, including viral mimics of miR- 155, miR-142-3p and miR-23. Recently, we noticed an extended, but offset, sequence similarity between the KSHV miRNA miR-K6-5p and the tumor suppressor cellular miR-16. Here, we have characterized the relationship between miR-K6-5p and miR-16. RNA- sequencing and extensive validation experiments show that miR-K6-5p and miR-16 both use canonical seed matching for target regulation. At the target level, miR-K6-5p was able to regulate most miR-16 targets, in some instances at altered efficiencies compared to miR-16. Additionally, miR-K6-5p regulates a small number of targets that are not shared with miR-16. At the functional level, miR-K6-5p shared tumor suppressive functions of miR- 16, including the induction of cell cycle arrest. Altogether, our data suggest that this oncogenic herpesvirus encodes a functional mimic of miR-16. Our ongoing experiments address the hypothesis that miR-K6-5p functions to balance consequences of viral oncogene expression. While many oncogenic herpesviruses encode gene products that antagonize tumor suppressors, this is – to our knowledge – the first example of an oncogenic virus that encodes a homolog or mimic of a bona fide tumor suppressor.
Partial cyclin derives proliferation
Viral FLIP inhibit apoptosis/porters survival
LANA maintains genome inhibits, p53
~20 viral miRNAs
Immunodeficiency —> B cell lymphoma or KS
MiR-K6-5p has extended sequence similarity to cellular miR-15/16
- miR-15/16 are well established tumor suppressors
- G1/S cycling CCND1, 2, 3, E1
- CDKs
- Inhibitors of BCL2, MCL1
- 13q deletion in cancer patients
MiR16 and miR-K6-5p have shared and unique seed-based target
- overlapping extensive canonical sites result in mimicry
- MiR-K6-5p and miR-16 both cause cell cycle arrest
Balance between
Viral oncogenes (vCyclin, miR-). Viral suppressors (miR-K6-5p)
Oral Talk #80
Role of mTOR Complexes in the Maintenance of
Nasopharyngeal Carcinoma Cancer Stem Cells
Nannan Zhu1, Qian Wang1, Yan Wang1, Yan Yuan1,2
1Institute of Human Virology, Sun Yat-sen University, 2 Department of Microbiology, University of Pennsylvania School of Dental Medicine
Epstein-Barr Virus (EBV) is associated with several malignant diseases including nasopharyngeal carcinoma (NPC), certain lymphomas, and a portion of gastric cancers. Virus-encoded oncoproteins LMP1 and LMP2A have been shown to induce the epithelial- to-mesenchymal transition (EMT) and increase the stemness of NPC. We and others have found that LMP1 and LMP2A activate PI3K/mTOR/Akt signaling pathway, resulting in an increased number of cancer stem cells (CSCs), while inhibition of mTORC1 and mTORC2 by mTOR dual inhibitor preferentially diminished CSCs levels. These observations suggest mTORC1/2/Akt pathway is essential for NPC to maintain CSCs. Since CSCs have been implicated in cancer recurrence, metastasis, and therapeutic resistance, understanding of how CSCs are generated and maintained in NPC will lead to effective targeting strategies for NPC CSCs and novel treatment of NPC. Towards this goal, we investigated how EBV- activated mTOR pathway contributes to generation and maintenance of CSCs in NPC and underlying mechanisms. First, using an shRNA-mediated knockdown approach, we demonstrated an essential role of mTORC2 in NPC tumor initiation capacity, as silencing the expression of Rictor (an essential component of mTORC2) completely abolished NPC tumor sphere formation. Second, mTORC1 is important for NPC therapeutic resistance, as knockdown of Raptor expression (a critical component of mTORC1) markedly inhibited side population in NPC. Third, both mTORC1 and mTORC2 are required to regulate NPC migration and invasion, suggesting that mTORC1/2 co-regulate metastasis of NPC. In addition, mTORC2 down-regulated the expression of SNAI1 which represses E-cadherin gene transcription and inhibited EMT-associated cytoskeletal change, while mTORC1 activates the expression of EMT-associated transcriptions factors BMI-1, KLF-4, and c- myc. Taken together, effective targeting of NPC CSCs requires inhibition of both mTORC1 and mTORC2 to achieve durable remission of NPC in patients. This notion has been approved by our result that an mTOR dual inhibitor (NVP-BEZ235) delayed tumor growth in NPC xenograft models.
Nasopharyngeal Carcinoma Cancer Stem Cells
Nannan Zhu1, Qian Wang1, Yan Wang1, Yan Yuan1,2
1Institute of Human Virology, Sun Yat-sen University, 2 Department of Microbiology, University of Pennsylvania School of Dental Medicine
Epstein-Barr Virus (EBV) is associated with several malignant diseases including nasopharyngeal carcinoma (NPC), certain lymphomas, and a portion of gastric cancers. Virus-encoded oncoproteins LMP1 and LMP2A have been shown to induce the epithelial- to-mesenchymal transition (EMT) and increase the stemness of NPC. We and others have found that LMP1 and LMP2A activate PI3K/mTOR/Akt signaling pathway, resulting in an increased number of cancer stem cells (CSCs), while inhibition of mTORC1 and mTORC2 by mTOR dual inhibitor preferentially diminished CSCs levels. These observations suggest mTORC1/2/Akt pathway is essential for NPC to maintain CSCs. Since CSCs have been implicated in cancer recurrence, metastasis, and therapeutic resistance, understanding of how CSCs are generated and maintained in NPC will lead to effective targeting strategies for NPC CSCs and novel treatment of NPC. Towards this goal, we investigated how EBV- activated mTOR pathway contributes to generation and maintenance of CSCs in NPC and underlying mechanisms. First, using an shRNA-mediated knockdown approach, we demonstrated an essential role of mTORC2 in NPC tumor initiation capacity, as silencing the expression of Rictor (an essential component of mTORC2) completely abolished NPC tumor sphere formation. Second, mTORC1 is important for NPC therapeutic resistance, as knockdown of Raptor expression (a critical component of mTORC1) markedly inhibited side population in NPC. Third, both mTORC1 and mTORC2 are required to regulate NPC migration and invasion, suggesting that mTORC1/2 co-regulate metastasis of NPC. In addition, mTORC2 down-regulated the expression of SNAI1 which represses E-cadherin gene transcription and inhibited EMT-associated cytoskeletal change, while mTORC1 activates the expression of EMT-associated transcriptions factors BMI-1, KLF-4, and c- myc. Taken together, effective targeting of NPC CSCs requires inhibition of both mTORC1 and mTORC2 to achieve durable remission of NPC in patients. This notion has been approved by our result that an mTOR dual inhibitor (NVP-BEZ235) delayed tumor growth in NPC xenograft models.
MTOR maintains the proliferation of NPC CSC cells
MTOR inhibitor CSC27 from Tradutinal Herb Medicine specifically inhibitors AKT Ser472, altering the subscellular localization of MTOR , NPC tumor formation in mice (BEZ2345 is a control)
Oral Talk #81
An Essential Role of Urea Cycle and Argininosuccinate
Synthase 1 in KSHV-Induced Cellular Transformation
TIngting Li1, Shou-Jiang Gao1
1University of Southern California
Cancer cells are required to rewire existing metabolic pathways for efficient uptake and incorporation of nutrients to support their anabolic growth. We have previously shown that, unlike most other cancer cells that are addicted to glucose and aerobic glycolysis (Warburg effect), Kaposi’s sarcoma-associated herpesvirus (KSHV)-transformed cells do not depend on glucose and have a reduced level of aerobic glycolysis. Instead, KSHV-transformed cells are addicted to glutamine, which is shunted for the synthesis of amino acids and nucleotides in addition to energy production. It has been reported that high level consumption of glutamine by cells requires tight regulation and timely clearance of excess nitrogen to avoid accumulation of toxic byproducts, which can be achieved by the urea cycle. Indeed, we have found that KSHV infection accelerates the urea cycle by upregulating argininosuccinate synthase 1 (ASS1), a key enzyme in the urea cycle. KSHV upregulation of ASS1 requires the oncogenic viral miRNAs. Knockdown of ASS1 suppresses cell proliferation and abolishes colony formation in soft agar of KSHV- transformed cells. Furthermore, ASS1 is required for KSHV activation of the STAT3 pathway, an essential pathway required for KSHV-induced abnormal cell proliferation and cellular transformation. These results illustrate a novel mechanism by which an oncogenic virus hijacks a key metabolic pathway to promote cellular transformation and reveal a potential novel therapeutic target for KSHV-induced malignancies.
Synthase 1 in KSHV-Induced Cellular Transformation
TIngting Li1, Shou-Jiang Gao1
1University of Southern California
Cancer cells are required to rewire existing metabolic pathways for efficient uptake and incorporation of nutrients to support their anabolic growth. We have previously shown that, unlike most other cancer cells that are addicted to glucose and aerobic glycolysis (Warburg effect), Kaposi’s sarcoma-associated herpesvirus (KSHV)-transformed cells do not depend on glucose and have a reduced level of aerobic glycolysis. Instead, KSHV-transformed cells are addicted to glutamine, which is shunted for the synthesis of amino acids and nucleotides in addition to energy production. It has been reported that high level consumption of glutamine by cells requires tight regulation and timely clearance of excess nitrogen to avoid accumulation of toxic byproducts, which can be achieved by the urea cycle. Indeed, we have found that KSHV infection accelerates the urea cycle by upregulating argininosuccinate synthase 1 (ASS1), a key enzyme in the urea cycle. KSHV upregulation of ASS1 requires the oncogenic viral miRNAs. Knockdown of ASS1 suppresses cell proliferation and abolishes colony formation in soft agar of KSHV- transformed cells. Furthermore, ASS1 is required for KSHV activation of the STAT3 pathway, an essential pathway required for KSHV-induced abnormal cell proliferation and cellular transformation. These results illustrate a novel mechanism by which an oncogenic virus hijacks a key metabolic pathway to promote cellular transformation and reveal a potential novel therapeutic target for KSHV-induced malignancies.
Why ASS1
- KSHV transformed cells rely on glutamine, rather tha glucose
- Hypothesis: KSHV proteins
ASS1 is unregulated in multiple KSHV infected
Viral latent products expressed during KSHV latency
- Deletion mutants of latent genes
- Turns out miRNAs are responsible for ASS1 up-regulation
- ASS1 KO induces cell cycle arrest and apoptosis
- INOS is unregulated by KSHV transformed cells
- ASS1 KO reduces intravenously NO and inactivates STAT3
- TLR4 mediated inflammation promotes KSHV-induced ASS1 unregulation
- NO don’t SNAP partially rescues STAT2 activation induced by ASd1 KO
- ASS1 KO leads to decreased STAT2 activation , which I is in part mediated
Oral Talk #82
Reverse Engineering EBV Driven B Cell Transformation and
Lymphomagenesis in the Mouse
Thomas Sommermann1, Jonathan Ronen1, Klaus Rajewsky1
1MDC – Max Delbrueck Center for Molecular Medicine
EBV has the intrinsic capacity to transform human B cells into continuously proliferating lymphoblastoid cell lines (LCL). In healthy individuals EBV-driven B cells are under tight immune control, while immunosuppressed patients often suffer from EBV-positive B cell lymphoproliferative diseases (LPD) and lymphomas. Using transgenic mouse models, we had shown before that EBV Latent membrane protein 1 (LMP1) is sufficient to induce mouse B cell proliferation. In immunosuppressed (T cell deficient) mice, LMP1-expressing B cells caused LPD but needed additional mutations to transform into B cell lymphomas. We now defined one of these secondary hits as the aberrant activation of Early B cell factor 1 (EBF1). EBF1 over expression is sufficient to fully transform LMP1 expressing mouse B cells in vitro into lymphoblastoid cell lines and in vivo into malignant tumors. LMP1 expression alone (or even more in the combination with LMP2A) pushes mouse B cells towards plasma cell differentiation, while LMP-driven lymphomagenesis requires a block of such terminal differentiation. We find EBF1 to be a potent inhibitor of LMP-induced plasma cell differentiation, implying repression of plasma cell differentiation as a key event downstream of EBF1 activation in LMP-driven lymphomagenesis.
As EBF1 is not reported to be aberrantly activated in human EBV-driven B cell tumors, we speculated EBV itself encodes gene products that suppress plasma cell differentiation. Indeed, B cells of Epstein Barr nuclear antigen 3A (EBNA3A) transgenic mice do not differentiate to plasma cells. In B cells of EBNA3A und LMP double-transgenic mice EBNA3A suppresses LMP-induced plasma cell differentiation and promotes lymphomagenesis.
Taken together, our findings highlight LMP-induced plasma cell differentiation as a crucial pathway that must be inhibited either by activation of EBF1 or expression of EBNA3A in order to sustain proliferation and transformation of EBV-infected B cells.
Lymphomagenesis in the Mouse
Thomas Sommermann1, Jonathan Ronen1, Klaus Rajewsky1
1MDC – Max Delbrueck Center for Molecular Medicine
EBV has the intrinsic capacity to transform human B cells into continuously proliferating lymphoblastoid cell lines (LCL). In healthy individuals EBV-driven B cells are under tight immune control, while immunosuppressed patients often suffer from EBV-positive B cell lymphoproliferative diseases (LPD) and lymphomas. Using transgenic mouse models, we had shown before that EBV Latent membrane protein 1 (LMP1) is sufficient to induce mouse B cell proliferation. In immunosuppressed (T cell deficient) mice, LMP1-expressing B cells caused LPD but needed additional mutations to transform into B cell lymphomas. We now defined one of these secondary hits as the aberrant activation of Early B cell factor 1 (EBF1). EBF1 over expression is sufficient to fully transform LMP1 expressing mouse B cells in vitro into lymphoblastoid cell lines and in vivo into malignant tumors. LMP1 expression alone (or even more in the combination with LMP2A) pushes mouse B cells towards plasma cell differentiation, while LMP-driven lymphomagenesis requires a block of such terminal differentiation. We find EBF1 to be a potent inhibitor of LMP-induced plasma cell differentiation, implying repression of plasma cell differentiation as a key event downstream of EBF1 activation in LMP-driven lymphomagenesis.
As EBF1 is not reported to be aberrantly activated in human EBV-driven B cell tumors, we speculated EBV itself encodes gene products that suppress plasma cell differentiation. Indeed, B cells of Epstein Barr nuclear antigen 3A (EBNA3A) transgenic mice do not differentiate to plasma cells. In B cells of EBNA3A und LMP double-transgenic mice EBNA3A suppresses LMP-induced plasma cell differentiation and promotes lymphomagenesis.
Taken together, our findings highlight LMP-induced plasma cell differentiation as a crucial pathway that must be inhibited either by activation of EBF1 or expression of EBNA3A in order to sustain proliferation and transformation of EBV-infected B cells.
Transgenic knock-in mice allows tightly controlled expression of EBV trans genes
ROSA26
C19CreB
LMP1 expressing B cells are controlled by T cells (Zhang, Yasuda, Cell 2012)
- CD19cre in B cells LMP expression
- EBF1 is recurrently translocated in to the IgH locus (chr 12 113,12)
- EBF1 translocation artificially induces EBF1 expression
- EBF1 expression is sufficient to transform LMP1 in B cells
EBF1 is B cell identify
Activation of NFkB and inhibition of plasma cell differentiation
EBF1 blocks LMP1-mediated induction of plasma cell differentiation in LMP1 driven B cells blasts and tumors
EBF1 blocks LMP1 driven plasma cell differentiation and allows transformation
Combination of LMP1 and EBNA3A
EBNA3A blocks B cell to plasma cell differentiation
CD19cre GFP
Should check on EBF1 in NPC cells!
Oral Talk #83
An EBNA3C-Deleted Epstein-Barr Virus (EBV) Mutant Causes
B-Cell Lymphomas with Delayed Onset in a Cord Blood-
Humanized Mouse Model
James Romero-Masters1, Makoto Ohashi1, Reza Djavadian1, Mark Eichelberg1, Jillian Bristol1, Shidong Ma1, Erik Ranheim1, Jenny Gumperz1, Eric Johannsen1, Shannon Kenney1
1University of Wisconsin-Madison School of Medicine and Public Health, University of Wisconsin- Madison
EBV causes human B-cell lymphomas and transforms B cells in vitro. EBNA3C, an EBV protein expressed in latently-infected cells, is required for EBV transformation of B cells in vitro. However, many EBV+ lymphomas do not express this protein, suggesting that it may be less important in vivo or that cellular mutations and/or signaling pathways may obviate the need for EBNA3C. EBNA3C collaborates with EBNA3A to repress expression of the CDKN2A-encoded tumor suppressors, p16 and p14, and EBNA3C deleted EBV transforms B cells containing a p16 germline mutation in vitro. Here we have examined the phenotype of an EBNAC-deleted virus (Δ3C) in a cord blood-humanized mouse model (CBH). We found that the Δ3C virus induced fewer lymphomas (occurring with a delayed onset) in comparison to the wild-type (WT) control virus, although a subset (10/26) of Δ3C-infected CBH mice eventually developed invasive diffuse large B cell lymphomas with type III latency. Both WT and Δ3C viruses induced monoclonal B-cell lymphomas (without somatic hyper-mutation) that were infiltrated by polyclonal T cells. In comparison to WT-infected tumors, Δ3C-infected tumors had greatly increased p16 levels, and RNA-seq analysis revealed a decrease in E2F target gene expression. However, we found that Δ3C-infected tumors expressed c-Myc and cyclin E at similar levels compared to WT-infected tumors, allowing cells to at least partially bypass p16-mediated cell cycle inhibition. The anti- apoptotic proteins, BCL2 and IRF4, were expressed in Δ3C-infected tumors, while the pro- apoptotic protein, BIM, was minimally expressed (likely helping cells avoid c-Myc-induced apoptosis). Unexpectedly, Δ3C-infected tumors had increased T-cell infiltration, increased expression of T-cell chemokines (CCL5, CCL20 and CCL22) and enhanced type I interferon response in comparison to WT tumors. Together, these results reveal that EBNA3C contributes to, but is not essential for, EBV-induced lymphomagenesis in CBH mice, and suggest potentially important immunologic roles of EBNA3C in vivo.
B-Cell Lymphomas with Delayed Onset in a Cord Blood-
Humanized Mouse Model
James Romero-Masters1, Makoto Ohashi1, Reza Djavadian1, Mark Eichelberg1, Jillian Bristol1, Shidong Ma1, Erik Ranheim1, Jenny Gumperz1, Eric Johannsen1, Shannon Kenney1
1University of Wisconsin-Madison School of Medicine and Public Health, University of Wisconsin- Madison
EBV causes human B-cell lymphomas and transforms B cells in vitro. EBNA3C, an EBV protein expressed in latently-infected cells, is required for EBV transformation of B cells in vitro. However, many EBV+ lymphomas do not express this protein, suggesting that it may be less important in vivo or that cellular mutations and/or signaling pathways may obviate the need for EBNA3C. EBNA3C collaborates with EBNA3A to repress expression of the CDKN2A-encoded tumor suppressors, p16 and p14, and EBNA3C deleted EBV transforms B cells containing a p16 germline mutation in vitro. Here we have examined the phenotype of an EBNAC-deleted virus (Δ3C) in a cord blood-humanized mouse model (CBH). We found that the Δ3C virus induced fewer lymphomas (occurring with a delayed onset) in comparison to the wild-type (WT) control virus, although a subset (10/26) of Δ3C-infected CBH mice eventually developed invasive diffuse large B cell lymphomas with type III latency. Both WT and Δ3C viruses induced monoclonal B-cell lymphomas (without somatic hyper-mutation) that were infiltrated by polyclonal T cells. In comparison to WT-infected tumors, Δ3C-infected tumors had greatly increased p16 levels, and RNA-seq analysis revealed a decrease in E2F target gene expression. However, we found that Δ3C-infected tumors expressed c-Myc and cyclin E at similar levels compared to WT-infected tumors, allowing cells to at least partially bypass p16-mediated cell cycle inhibition. The anti- apoptotic proteins, BCL2 and IRF4, were expressed in Δ3C-infected tumors, while the pro- apoptotic protein, BIM, was minimally expressed (likely helping cells avoid c-Myc-induced apoptosis). Unexpectedly, Δ3C-infected tumors had increased T-cell infiltration, increased expression of T-cell chemokines (CCL5, CCL20 and CCL22) and enhanced type I interferon response in comparison to WT tumors. Together, these results reveal that EBNA3C contributes to, but is not essential for, EBV-induced lymphomagenesis in CBH mice, and suggest potentially important immunologic roles of EBNA3C in vivo.
EBNA3C and 3A:
Co-opt with other signaling pathways in inactivating p16
- LMP1-CD40
- LMP2-BCR
- EBNA2 – NOTCH
Delta EBNA3C induced tumors overexpress p16 in comparison with WT EBNA3, but still express MYC and .. in huNSG
Delta3C induced lymphoma has subset of BIM overexoresuion
Delta increased T cell infiltration
WT and delta3C induced lymphoma halve monolonal oligoclonal B cell populations
- RNA-seq analysis. Down of cell cycle genes (consistent with slower cell proliferationO
- Up with immune genes (qPCR reveals that her there is an increase I
- CD8A, GZMB, PFR1, CCL20, CCL5 and CCL22
- CCL5 is positively in the 3C deleted lymphoma
- EBNA2C may inhibit the type 1 interferon and immune response
- Highly