We have focused in three related areas: the study of vFLIP, a KSHV latent gene product expressed in Kaposi's sarcoma (KS), Primary Effusion Lymphoma (PEL) and multicentric Castleman's disease;the study of CXCR7, a G protein-coupled receptor induced by KSHV in the host cells;and the development of new therapies for KSHV-induced malignances occurring in AIDS patients. One of the characteristic features of KSHV is its ability to promote angiogenesis and lymphangiogenesis. Unlike other viruses that have hijacked cellular genes that promote angiogenes, KHSV does not code for pro-angiogenic factors but rather promotes the expression of pro-angiogenic genes by the cells it infects. ORFK13/vFLIP encodes a 188-amino acid protein, which binds to the Iκb kinase (IKK) complex to activate NFκB. 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 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. The microarray analysis found that ORFK13/vFLIP promotes the expression of thymidine phosphorylase, a cellular enzyme that can metabolize the prodrug 5-fluoro-5-deoxyuridine (5-dFUrd) to 5-fluouridine (5-FU). A potent thymidine synthase inhibitor, 5-Fu blocks DNA and RNA synthesis. When tested for cytotoxicity, 5-dFUrd (0.1-1μM) selectively killed ORFK13/vFLIP-expressing endothelial cells while sparing control cells. These results demonstrate that ORFK13/vFLIP directly and indirectly contributes to the inflammatory and vascular phenotype of KS, and identify 5-dFUrd as a potential new drug that targets KSHV latency for the treatment of KS and other KSHV-associated malignancies. One of the cellular genes that are highly induced by KSHV is the chemokine receptor RDC1/CXCR7. Recent studies have shown that CXCR7 binds the chemokines SDF1 and MCP1 but it is still unclear whether CXCR7 can signal in response to these ligands or other signals, or whether its function is to serve to sequester ligands away from their receptors. Recently, CXCR7 was shown to oligomerize with CXCR4, a receptor that can signals in response to SDF1. We are interested in the function of CXCR7 in the context of KSHV infection. Initial observations in mice have shown that CXCR7 promotes KSHV-induced tumor progression, but the mechanisms underlying this effect are currently being investigated. Primary effusion lymphoma (PEL) is a fatal viral malignancy, which typically presents as a malignant effusion that later disseminates. In spite of therapy with high-dose chemotherapy or other therapies, PEL is a rapidly fatal malignancy. Rapamycin, which targets mTOR (mammalian target of rapamycin), an effector of cell signaling pathways often deregulated in cancer, showed efficacy against a variety of tumors, particularly those of lymphoid originin. We have investigated the potential utility of Rapamycin for the treatment of experimental PEL. Previous studies have suggested that rapamycin could be effective against subcutaneous PEL in mice. However, this pre-clinical model is far removed from the disease in patients in its location and progression. Recently, PEL development in rapamycin-treated post-transplant recipients raised questions about the drugs anti-PEL activity. We have developed and used a murine model of effusion PEL progressing to peritoneal tumors to investigate the anti-PEL activity of rapamycin. We found that rapamycin significantly reduces ascites accumulation and extends mouse survival. Initially, rapamycin reduced PEL load compared to control mice, but most mice rapidly showed PEL progression. Levels of VEGF, which promotes vascular permeability contributing to effusion formation, were significantly reduced in ascites of rapamycin-treated mice compared to controls. Expression of IL-10, the principal autocrine growth factor for PEL, was initially reduced in PEL from rapamycin-treated mice but rapidly increased despite treatment. We found that the hypoxic environment of ascites and rapamycin cooperate in stimulating IL-10 expression in PEL. These results do not support the use of rapamycin as a curative treatment for PEL, but identify rapamycin an effective drug to reduce accumulation of malignant effusions. Current efforts in the laboratory are intended to further characterize development of PEL resistance to rapamycin and how to prevent it.
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