Gianna Ballon

NF-κB signaling modulation by EBV and KSHV

The nuclear factor (NF)-kappaB signaling pathway is pivotal for immune system function. Not surprisingly, pathogenic microorganisms have developed strategies to subvert it. Two examples are Epstein-Barr virus (EBV) and Kaposi sarcoma herpesvirus (KSHV), oncogenic gammaherpesviruses that establish a lifelong latent infection in their human hosts. The modulation of NF-kappaB signaling by EBV and KSHV is not only important for viral infection, but also contributes to the development of malignant neoplasia. This review explores the current knowledge of NF-kappaB modulation by EBV and KSHV, focusing on connections between viral biology and human carcinogenesis.

DNA methyltransferase 1 and DNA methylation patterning contribute to germinal center B-cell differentiation.

The phenotype of germinal center (GC) B cells includes the unique ability to tolerate rapid proliferation and the mutagenic actions of activation induced cytosine deaminase (AICDA). Given the importance of epigenetic patterning in determining cellular phenotypes, we examined DNA methylation and the role of DNA methyltransferases in the formation of GCs. DNA methylation profiling revealed a marked shift in DNA methylation patterning in GC B cells versus resting/naive B cells. This shift included significant differential methylation of 235 genes, with concordant inverse changes in gene expression affecting most notably genes of the NFkB and MAP kinase signaling pathways. GC B cells were predominantly hypomethylated compared with naive B cells and AICDA binding sites were highly overrepresented among hypomethylated loci. GC B cells also exhibited greater DNA methylation heterogeneity than naive B cells. Among DNA methyltransferases (DNMTs), only DNMT1 was significantly up-regulated in GC B cells. Dnmt1 hypomorphic mice displayed deficient GC formation and treatment of mice with the DNA methyltransferase inhibitor decitabine resulted in failure to form GCs after immune stimulation. Notably, the GC B cells of Dnmt1 hypomorphic animals showed evidence of increased DNA damage, suggesting dual roles for DNMT1 in DNA methylation and double strand DNA break repair.

Kaposi sarcoma herpesvirus (KSHV) vFLIP oncoprotein induces B cell transdifferentiation and tumorigenesis in mice

Kaposi sarcoma herpesvirus (KSHV) is specifically associated with Kaposi sarcoma (KS) and 2 B cell lymphoproliferative diseases, namely primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). KS, PEL, and MCD are largely incurable and poorly understood diseases most common in HIV-infected individuals. Here, we have revealed the role of viral FLICE-inhibitory protein (vFLIP) in the initiation of PEL and MCD by specifically expressing vFLIP at different stages of B cell differentiation in vivo. Mice showed MCD-like abnormalities and immunological defects including lack of germinal centers (GCs), impaired Ig class switching, and affinity maturation. In addition, they showed increased numbers of cells expressing cytoplasmic IgM-λ, a thus far enigmatic feature of the KSHV-infected cells in MCD. B cell-derived tumors arose at high incidence and displayed Ig gene rearrangement with downregulated expression of B cell-associated antigens, which are features of PEL. Interestingly, these tumors exhibited characteristics of transdifferentiation and acquired expression of histiocytic/dendritic cell markers. These results define immunological functions for vFLIP in vivo and reveal what we believe to be a novel viral-mediated tumorigenic mechanism involving B cell reprogramming. Additionally, the robust recapitulation of KSHV-associated diseases in mice provides a model to test inhibitors of vFLIP as potential anticancer agents.

Targeting the Hsp90-associated viral oncoproteome in gammaherpesvirus-associated malignancies.

PU-H71 is a purine-scaffold Hsp90 inhibitor that, in contrast to other Hsp90 inhibitors, displays unique selectivity for binding the fraction of Hsp90 that is preferentially associated with oncogenic client proteins and enriched in tumor cells (teHsp90). This property allows PU-H71 to potently suppress teHsp90 without inducing toxicity in normal cells. We found that lymphoma cells infected by Epstein-Barr virus or Kaposi sarcoma-associated herpes virus (KSHV) are exquisitely sensitive to this compound. Using PU-H71 affinity capture and proteomics, an unbiased approach to reveal oncogenic networks, we identified the teHsp90 interactome in KSHV(+) primary effusion lymphoma cells. Viral and cellular proteins were identified, including many involved in nuclear factor (NF)-κB signaling, apoptosis, and autophagy. KSHV vFLIP is a viral oncoprotein homologous to cFLIPs, with NF-κB-activating and antiapoptotic activities. We show that teHsp90 binds vFLIP but not cFLIPs. Treatment with PU-H71 induced degradation of vFLIP and IKKγ, NF-κB downregulation, apoptosis and autophagy in vitro, and more importantly, tumor responses in mice. Analysis of the interactome revealed apoptosis as a central pathway; therefore, we tested a BCL2 family inhibitor in primary effusion lymphoma cells. We found strong activity and synergy with PU-H71. Our findings demonstrate PU-H71 affinity capture identifies actionable networks that may help design rational combinations of effective therapies.

The tumor virus landscape of AIDS-related lymphomas

Immunodeficiency dramatically increases susceptibility to cancer as a result of reduced immune surveillance and enhanced opportunities for virus-mediated oncogenesis. Although AIDS-related lymphomas (ARLs) are frequently associated with known oncogenic viruses, many cases contain no known transforming virus. To discover novel transforming viruses, we profiled a set of ARL samples using whole transcriptome sequencing. We determined that Epstein-Barr virus (EBV) was the only virus detected in the tumor samples of this cohort, suggesting that if unidentified pathogens exist in this disease, they are present in <10% of cases or undetectable by our methods. To evaluate the role of EBV in ARL pathogenesis, we analyzed viral gene expression and found highly heterogeneous patterns of viral transcription across samples. We also found significant heterogeneity of viral antigen expression across a large cohort, with many patient samples presenting with restricted type I viral latency, indicating that EBV latency proteins are under increased immunosurveillance in the post-combined antiretroviral therapies era. Furthermore, EBV infection of lymphoma cells in HIV-positive individuals was associated with a distinct host gene expression program. These findings provide insight into the joint host-virus regulatory network of primary ARL tumor samples and expand our understanding of virus-associated oncogenesis. Our findings may also have therapeutic implications, as treatment may be personalized to target specific viral and virus-associated host processes that are only present in a subset of patients.

A20 (TNFAIP3) genetic alterations in EBV-associated AIDS-related lymphoma

A20, a negative regulator of NF-κB, has been implicated as a tumor suppressor gene in multiple types of B-cell lymphoma. AIDS-related lymphomas (ARLs) are high-grade B-cell lymphomas that are frequently associated with EBV infection. We examined a panel of ARLs for A20 alterations. FISH showed A20 deletion in 6 of 33 cases (18%). A20 mutations were found in 3 of 19 cases (16%), including 2 cases with deletions of the comple-mentary allele. Immunohistochemistry showed the absence of A20 protein in 7 of 55 samples (13%). In contrast to reports in Hodgkin lymphoma in which EBV infection and A20 alteration are mutually exclusive, A20 inactivation was observed in both EBV(+) and EBV(-) cases. The EBV latent membrane protein 1, which activates NF-κB, was not expressed in 12 of 13 cases with A20 loss. In ARLs loss of A20 may be an alternative mechanism of NF-κB activation in the absence of latent membrane protein 1 expression.

Systemic Expression of Kaposi Sarcoma Herpesvirus (KSHV) Vflip in Endothelial Cells Leads to a Profound Proinflammatory Phenotype and Myeloid Lineage Remodeling In Vivo

KSHV is the causative agent of Kaposi sarcoma (KS), a spindle-shaped endothelial cell neoplasm accompanied by an inflammatory infiltrate. To evaluate the role of KSHV vFLIP in the pathogenesis of KS, we constructed mice with inducible expression of vFLIP in endothelial cells. Abnormal cells with endothelial marker expression and fusiform appearance were observed in several tissues reminiscent of the spindle cells found in KS. Serum cytokines displayed a profound perturbation similar to that described in KSHV inflammatory cytokine syndrome (KICS), a recently described clinical condition characterized by elevated IL6 and IL10. An increased myeloid component with suppressive immune phenotype was found, which may contribute to functional changes in the microenvironment and cellular heterogeneity as observed in KS. These mice represent the first in vivo demonstration that vFLIP is capable of inducing vascular abnormalities and changes in host microenvironment with important implications for understanding the pathogenesis and treating KSHV-associated diseases.

Abstract 4829: A purine scaffold Hsp90 inhibitor has antitumor activity in KSHV-associated malignancies by suppressing vFLIP

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Kaposis sarcoma herpesvirus (KSHV/ HHV-8), a member of the α-herpesvirus family of human DNA viruses, is the etiologic agent of several malignancies in immune-compromised individuals, such as Kaposis sarcoma (KS) and primary effusion lymphoma (PEL). Activated NF-κB is a critical mechanism by which KSHV-infected PEL cells are protected from apoptosis. KSHV vFLIP has been identified as a viral oncogene that is responsible for NF-κB-dependent anti-apoptotic gene expression in PEL cells, and also prevents autophagy in KSHV-infected endothelial cells. In particular, the IKK signaling complex consisting of IKKα, IKKα, IKKα, vFLIP and Hsp90 was shown to be essential for survival in PEL cells. The chaperone protein Hsp90 binds client proteins involved in the regulation of cell survival and apoptosis signal transduction, including Akt, IKK complex, and KSHV vFLIP. A lack of Hsp90 causes protein misfolding, ubiquitination and degradation. The Hsp90 inhibitor geldanamycin was previously tested in PEL cells and shown to inhibit activity of the IKK complex in vitro. However, geldanamycin is of limited therapeutic potential due to its undesirable pharmacophysiology. We tested a new purine-scaffold Hsp90 inhibitor with high selectivity for tumor versus normal cell Hsp90, which is water-soluble with high oral bioavailability and excellent therapeutic window. Materials and methods: We evaluated the sensitivity of several KSHV infected and uninfected cell lines to treatment with this inhibitor, designated PU-H71. We performed viability and NF-κB reporter luciferase assays, and immunoblot and qRT-PCR analyses of cellular and viral proteins. Finally, we used a mouse PEL xenograft model and in vivo imaging to assess tumor responses to PU-H71. Results: We found all KSHV-positive PEL cell lines to be exquisitely sensitive when compared to uninfected lymphoma cells, with LC50s in the nanomolar range. PU-H71 was shown to induce PEL cell death by apoptosis and autophagy within 48 hours of treatment. PU-H71 also showed preferential toxicity in an in vitro model of KS. Pulldown demonstrated association of the compound with the active vFLIP-IKKα signalosome. Western blot analysis indicated that the IKK signaling complex was destabilized, and components vFLIP and IKKα were degraded upon PU-H71 treatment, resulting in inhibition of NF-κB signaling, as confirmed by reporter luciferase assay. qRT-PCR and reporter analysis indicated low levels of concomitant lytic reactivation. PU-H71 was further shown to inhibit progression of tumor spread and confer a significant survival advantage (p<0.02) in a mouse xenograft model of PEL, with persistence of IKKα destabilization ex vivo. Conclusions: Our findings demonstrate that Hsp90 inhibition with PU-H71 leads to reduced vFLIP levels in KSHV-infected cells, and to tumor responses. Thus, PU-H71 is a promising targeted approach for the treatment of KS and PEL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4829. doi:1538-7445.AM2012-4829

Chronic intestinal inflammation in mice expressing viral Flip in epithelial cells

Viruses are present in the intestinal microflora and are currently discussed as a potential causative mechanism for the development of inflammatory bowel disease. A number of viruses, such as Human Herpesvirus-8, express homologs to cellular FLIPs, which are major contributors for the regulation of epithelial cell death. In this study we analyzed the consequences of constitutive expression of HHV8-viral FLIP in intestinal epithelial cells (IECs) in mice. Surprisingly, expression of vFlip disrupts tissue homeostasis and induces severe intestinal inflammation. Moreover vFlipIEC-tg mice showed reduced Paneth cell numbers, associated with excessive necrotic cell death. On a molecular level vFlip expression altered classical and alternative NFκB activation. Blocking of alternative NFκB signaling by deletion of Ikka in vivo largely protected mice from inflammation and Paneth cell loss induced by vFLIP. Collectively, our data provide functional evidence that expression of a single viral protein in IECs can be sufficient to disrupt epithelial homeostasis and to initiate chronic intestinal inflammation.

TNFAIP3(A20) genetic alterations in AIDS-related lymphomas

Background AIDS-related lymphomas (ARLs), which include Burkitt lymphoma and diffuse large B cell lymphoma (DLBCL), are a heterogeneous group of lymphoproliferative disorders that occur in the setting of HIV-mediated immune suppression. A subset of cases are associated with EpsteinBarr virus (EBV) infection. EBV expresses latent viral oncoproteins that constitutively activate the transcription factor NFB, a potent inducer of genes involved in promoting B cell survival and proliferation [1]. In immunocompetent individuals, lymphomas that are not associated with EBV can also display increased NFB activity, and recent reports have described mutations in regulators of NFB. One of the frequently mutated regulatory genes is TNFAIP3, which encodes A20, a ubiquitin modifying enzyme involved in the termination of NFB signaling. Mutations resulting in the inactivation of A20 have been found in a significant proportion of marginal zone lymphomas [2], classical Hodgkin’s lymphomas and primary mediastinal B cell lymphomas [3], and DLBCLs [4]. In ARL the role of NFB activation and the incidence of mutations in A20 have not been described.