We have focused in four related areas: 1. the study of vFLIP, a KSHV latent gene product expressed in KSHV-infected cell targets and in Kaposi's sarcoma (KS), Primary Effusion Lymphoma (PEL) and Multicentric Castleman's disease (MCD); 2. the study of vIL-6, a KSHV viral product with structural similarity to cellular IL-6, and its role in the pathogenesis of MCD; 3. Peculiar clinical presentations of MCD; 4. The role of body cavity microenvironment in the pathogenesis of PEL; and 5. Development of new therapies for KSHV-induced malignancies occurring in AIDS patients. One of the characteristic features of KSHV is its ability to infect endothelial cells, and to indirectly promote angiogenesis and lymphangiogenesis predominantly by promoting the recruitment of cells that produce pro-angiogenic factors and promoting the expression of pro-angiogenic genes by the cells it infects. ORFK13/vFLIP encodes a 188-amino acid protein, which binds to the Ikb kinase (IKK) complex to activate NFkB. We examined ORFK13/vFLIP contribution to KS phenotype and potential for therapeutic targeting. To this end, we have retrovirally transduced ORFK13/vFLIP into primary human endothelial cells and examined the contribution of this gene to KS phenotype. We found that ORFK13/vFLIP induces the spindle morphology distinctive of KS cells and promotes formation of abnormal vascular networks typical of the disorderly KS vasculature. Microarray analysis of gene expression in endothelial cells transduced with ORFK13/vFLIP detected increased expression of certain pro-inflammatory cytokines, chemokines, and interferon-responsive genes. This study represents the first comprehensive analysis of gene regulation by KSHV-vFLIP. As one might expect from stimulation of pro-inflammatory cytokines and chemokines, we found that ORFK13/vFLIP stimulates adhesion of inflammatory cells characteristic of KS lesions. In additional experiments, we found that KSHV K13 induces the expression of the NF-kB regulatory proteins A20, ABIN-1 and ABIN-3 in primary human microvascular endothelial cells, and that KS spindle cells express A20 in KS tissue. In reporter assays, A20 strongly impaired K13-induced NF-kB activation in 293T cells, but ABIN-1 and ABIN-3 did not. Thus, these results provide evidence that KSHV finely modulates NF-kB in the host cells by both promoting NF-kB activation (resulting in expression of inflammatory cytokines) and tempering this activation by promoting expression of the NF-kB repressor A20 (resulting in inhibition of cell death). Previous studies from our group characterized vIL-6 as an early lytic gene expressed by KSHV, homologous to the cellular IL-6 cytokine. Unlike cellular IL-6, we found that vIL-6 can directly bind and signal through the gp130/JAK-STAT pathway without a requirement for the cellular IL-6 receptor, whose expression is restricted to certain cell types. Since gp130 is a fairly ubiquitous protein, vIL-6, in contract to the cellular cytokine, can activate virtually all cells in the body. Previously, we have transduced vIL6 in NIH3T3 cells; when these cells were transferred into T-cell immunodeficient mice, they generated tumors at a significantly higher rate than control NIH3T3 cells and, importantly, the mice developed splenomegaly, hepatomegaly and plasmacytosis in many tissues. These features are common to patients with MCD. We have now produced transgenic mouse lines in which vIL-6 is ubiquitously expressed. These mice were found to exhibit vIL-6 serum levels comparable with those observed in KSHV-infected patients, to contain elevated amounts of phosphorylated STAT3 in spleen and lymph nodes, where vIL-6 was abundantly produced, and to spontaneously develop key features of human plasma cell-type MCD, including splenomegaly, multifocal lymphadenopathy, hypergammaglobulinemia, and plasmacytosis. Interestingly, the vIL-6 transgenic mice crossed into IL-6-deficient mice did not yield the MCD-like phenotype observed in IL-6-competent mice. This indicated that endogenous cellular IL-6 is a critical co-factor in the natural history of MCD. We have previously reported that vIL-6 can induce the expression of cellular IL-6 in vitro, and that vIL-6 and cellular IL-6 are often detected at abnormally high levels in patients with MCD. These observations suggest that human IL-6 plays an important role in the pathogenesis of KSHV-associated MCD. Thus, in collaboration with the HAMB group at NCI we have initiated a clinical trial exploring the utility of blocking cellular IL-6 in the treatment of MCD. In related studies, we have identified three patients who presented with severe symptoms of MCD and had a rapid expansion of circulating B-cells (44-81%) attributable to HHV-8 positive cells sharing the phenotype (IgMl, CD19+, CD20-, CD138-) of HHV-8-infected cells from MCD lesions. These patients displayed a very high HHV-8 viral load in blood (7 logs HHV-8 DNA copies/ml) and high levels of serum vIL-6, the viral homologue of human interleukin 6. Serum IL-6 and IL-10 were also abnormally elevated. Thus, we have expanded the spectrum of HHV-8-related plasmablastic lymphoproliferative disorders in HIV-infected patients to include HHV-8+ polyclonal B-cell lymphocytosis. Additionally, we have identified a group of patients who resemble MCD patients in their clinical manifestations and laboratory parameters, but do not meet the histological criteria of MCD and they have no lymphadenopathy and no histologic evidence of MCD. We have labeled this new syndrome as KICS, KSHV-associated inflammatory cytokine syndrome. In other experiments we have examined the biochemical basis for diversity of phenotype within KS cells that are KSHV-infected. Such diversity has created uncertainties on the origin of KS tumor cells. We have examined the possibility that KSHV infection promotes endothelial to mesenchymal transition (EndMT). This process of endothelial cell conversion into mesenchymal cells plays critical roles during development of the heart, and underlies certain forms of pathological organ fibrosis and tissue ossification. We found that KSHV is an inducer of EndMT. Upon KSHV infection, primary dermal microvascular endothelial cells lose expression of endothelial markers, acquire expression of mesenchymal markers, display new invasive and migratory properties, and exhibit increased survival. We discovered that canonical Notch signaling pathway and the Notch-induced transcription factors Slug and ZEB1 are deployed by KSHV to induce activation of EndMT, whereas the TGF-beta signaling pathway previously linked to EndMT, is not utilized. The KSHV-infected spindle cells within KS lesions display a complex phenotype with features of endothelial and mesenchymal cells, display evidence of Notch activity and express nuclear ZEB1, features compatible with KSHV-induced EndMT in vivo. These results show that KSHV utilizes the EndMT program to endow endothelial cells with invasiveness and resistance to death. Additionally, KSHV regulates expression of DLC1, which modulates NFkB activation and its downtream targer A20. Targeting Notch signaling emerges as a novel experimental approach to the treatment of KS. PEL is a devastating lymphoma associated with KSHV, often in conjunction with EBV, which typically presents as a liquid malignancy in the body cavities. This peculiar location has often been attributed to high level VEGF secretion by PEL cells, which promotes vascular permeability associated with the pathogenesis of effusions. However, we have considered the possibility that the peculiar site of PEL presentation may reflect peculiar pro-tumorigenic functions of this niche. Current studies have identified mesothelial-derived factors that critically contribute to PEL development and growth. Targeting such factors is a current focus of our research on PEL.

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National Cancer Institute (NCI)
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Sanchez-Martin, David; Uldrick, Thomas S; Kwak, Hyeongil et al. (2017) Evidence for a Mesothelial Origin of Body Cavity Effusion Lymphomas. J Natl Cancer Inst 109:
Agerbaek, Mette Ø; Pereira, Marina A; Clausen, Thomas M et al. (2017) Burkitt lymphoma expresses oncofetal chondroitin sulfate without being a reservoir for placental malaria sequestration. Int J Cancer 140:1597-1608
Hu, Duosha; Wang, Victoria; Yang, Min et al. (2016) Induction of Kaposi's Sarcoma-Associated Herpesvirus-Encoded Viral Interleukin-6 by X-Box Binding Protein 1. J Virol 90:368-78
Espígol-Frigolé, Georgina; Planas-Rigol, Ester; Ohnuki, Hidetaka et al. (2016) Identification of IL-23p19 as an endothelial proinflammatory peptide that promotes gp130-STAT3 signaling. Sci Signal 9:ra28
Chen, Inn-Inn; Caprioli, Arianna; Ohnuki, Hidetaka et al. (2016) EphrinB2 regulates the emergence of a hemogenic endothelium from the aorta. Sci Rep 6:27195
Polizzotto, Mark N; Uldrick, Thomas S; Wyvill, Kathleen M et al. (2016) Clinical Features and Outcomes of Patients With Symptomatic Kaposi Sarcoma Herpesvirus (KSHV)-associated Inflammation: Prospective Characterization of KSHV Inflammatory Cytokine Syndrome (KICS). Clin Infect Dis 62:730-738
Sakakibara, Shuhei; Tosato, Giovanna (2014) Contribution of viral mimics of cellular genes to KSHV infection and disease. Viruses 6:3472-86
Sakakibara, S; Espigol-Frigole, G; Gasperini, P et al. (2013) A20/TNFAIP3 inhibits NF-?B activation induced by the Kaposi's sarcoma-associated herpesvirus vFLIP oncoprotein. Oncogene 32:1223-32
Oksenhendler, Eric; Boutboul, David; Beldjord, Kheira et al. (2013) Human herpesvirus 8+ polyclonal IgM? B-cell lymphocytosis mimicking plasmablastic leukemia/lymphoma in HIV-infected patients. Eur J Haematol 91:497-503
Gasperini, Paola; Espigol-Frigole, Georgina; McCormick, Peter J et al. (2012) Kaposi sarcoma herpesvirus promotes endothelial-to-mesenchymal transition through Notch-dependent signaling. Cancer Res 72:1157-69

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