The Kaposi's sarcoma-associated herpesvirus (KSHV, also called human herpesvirus 8) has been linked to KS and primary effusion lymphoma (PEL) in immunocompromised individuals. We report that PEL cell lines have constitutive active alternative NF-kappa B pathway and demonstrate high-level expression of NF-kappa B2/p100 precursor and its processed subunit p52. To elucidate the mechanism of activation of the alternative NF-kappa B pathway in PEL cells, we have investigated the role of KSHV-encoded viral Fas-associated death domain-like IL- beta 1-converting enzyme inhibitory protein (vFLIP) K13. We demonstrate that stable expression of K13, but not other FLIPs, in a variety of cell lines constitutively up-regulates p100/NF-kappa B2 expression and leads to its processing into the p52 subunit. K13-induced up-regulation and processing of p100 critically depends on the I kappa B kinase (IKK)alpha/IKK1 subunit of the IKK complex, whereas IKK beta/IKK2, receptor-interacting protein, and NF-kappa B-inducing kinase are dispensable for this process. Silencing of endogenous K13 expression by siRNA inhibits p100 processing and cellular proliferation. Our results demonstrate for the first time, to our knowledge, that KSHV vFLIP K13 is required for the growth and proliferation of PEL cells and alternative NF-kappa B pathway plays a key role in this process. Therapeutic agents targeting the alternative NF-kappa B pathway may have a role in the treatment of KSHV-associated lymphomas.

Primary effusion lymphoma (PEL) is an aggressive type of non-Hodgkin lymphoma localized predominantly in body cavities. Kaposi’s sarcoma-associated herpes virus is the causative agent of PEL. PEL is an incurable malignancy and has extremely poor prognosis when treated with conventional chemotherapy. Immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide are FDA approved drugs for the treatment of various ailments. IMiDs display pronounced anti-proliferative effect against majority of PEL cell lines within their clinically achievable concentrations, by arresting cells at G0/G1 phase of cell-cycle, and without any induction of KSHV lytic-cycle reactivation. Although microarray examination of PEL cells treated with lenalidomide revealed activation of interferon (IFN) signaling, blocking the IFN pathway did not block the anti-PEL activity of IMiDs. The anti-PEL effects of IMiDs involved cereblon-dependent suppression of IRF4 and rapid degradation of IKZF1, but not IKZF3. Small hairpin-RNA (shRNA) mediated knockdown of MYC enhanced the cytotoxicity of IMiDs. Bromodomain and extraterminal domain (BET) proteins are epigenetic readers which perform a vital role in chromatin remodeling and transcriptional regulation. BRD4, a widely expressed transcriptional coactivator, belongs to BET family of proteins, which has been shown to co-occupy the super-enhancers associated with MYC. Specific BRD4 inhibitors were developed which suppress MYC transcriptionally. Lenalidomide displayed synergistic cytotoxicity with several structurally distinct BRD4 inhibitors (JQ-1, IBET151, and PFI-1). Furthermore, combined administration of lenalidomide and BRD4 inhibitor JQ-1 significantly increased the survival of PEL bearing NOD.SCID mice in an orthotopic xenograft model as compared to either agent alone. These results provide compelling evidence for clinical testing of IMiDs alone and in combination with BRD4 inhibitors for PEL.

Kaposi's sarcoma-associated herpes virus ͉ primary effusion lymphoma

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Post-transcriptional gene silencing by small interfering RNAs (siRNAs) is rapidly becoming a powerful tool for genetic analysis of mammalian cells. Delivery of siRNA into mammalian cells is usually achieved via the transfection of double-stranded oligonucleotides or plasmids encoding RNA polymerase III promoter-driven small hairpin RNA. Recently, retroviral vectors have been used for siRNA delivery, which overcome the problem of poor transfection efficiency seen with the plasmid-based systems. However, retroviral vectors have several limitations, such as the need for active cell division for gene transduction, oncogenic potential, low titers and gene silencing. In this report, we have adapted a commercially available adenoassociated virus (AAV) vector for siRNA delivery into mammalian cells. We demonstrate the ability of this modified vector to deliver efficiently siRNA into HeLa S3 cells and downregulate p53 and caspase 8 expression. Our results suggest that AAV-based vectors are efficient vectors for the delivery of siRNA into mammalian cells. Based on the known ability of these vectors to infect both dividing and nondividing cells, their use as a therapeutic tool for the delivery of siRNA deserves further study.

Primary effusion lymphoma (PEL) is an aggressive form of non-Hodgkin's B cell lymphoma associated with infection by Kaposi's sarcoma associated herpesvirus (KSHV). (+)-JQ1 and I-BET151 are two recently described novel small molecule inhibitors of BET bromodomain chromatin-associated proteins that have shown impressive preclinical activity in cancers in which MYC is over-expressed at the transcriptional level due to chromosomal translocations that bring the MYC gene under the control of a super-enhancer. PEL cells, in contrast, lack structural alterations in the MYC gene, but have deregulated Myc protein due to the activity of KSHV-encoded latent proteins. We report that PEL cell lines are highly sensitive to BET bromodomain inhibitors-induced growth inhibition and undergo G0/G1 cell-cycle arrest, apoptosis and cellular senescence, but without the induction of lytic reactivation, upon treatment with these drugs. Treatment of PEL cell lines with BET inhibitors suppressed the expression of MYC and resulted in a genome-wide perturbation of MYC-dependent genes. Silencing of BRD4 and MYC expression blocked cell proliferation and cell-cycle progression, while ectopic expression of MYC from a retroviral promoter rescued cells from (+)-JQ1-induced growth arrest. In a xenograft model of PEL, (+)-JQ1 significantly reduced tumor growth and improved survival. Taken collectively, our results demonstrate that the utility of BET inhibitors may not be limited to cancers in which genomic alterations result in extremely high expression of MYC and they may have equal or perhaps greater activity against cancers in which the MYC genomic locus is structurally intact and c-Myc protein is deregulated at the post-translational level and is only modestly over-expressed.

The human herpes virus 8 (HHV8)-encoded viral FLICE (Fas-associating protein with death domain-like interleukin-1-converting enzyme) inhibitory protein (vFLIP) is believed to protect cells against death receptor-mediated apoptosis. In the present study we demonstrate that expression of HHV8 vFLIP in a growth factor-dependent TF-1 leukemia cell line protects against growth factor withdrawal-induced apoptosis. Unlike vector-expressing cells, those expressing HHV8 vFLIP maintain their mitochondrial membrane potential upon withdrawal from growth factor and also exhibit a block in the activation of caspases. The protective effect of HHV8 vFLIP is associated with its ability to activate the nuclear factor-kappa B (NF-kappaB) pathway and is missing in the vFLIP encoded by equine herpes virus 2 that lacks this activity. Inhibition of the NF-kappaB pathway by IkappaB superrepressor, lactacystin, MG132, arsenic trioxide, and phenylarsine oxide reverse the protection against growth factor withdrawal-induced apoptosis conferred by HHV8 vFLIP. HHV8 vFLIP up-regulates the expression of Bcl-x(L), an antiapoptotic member of the Bcl2 family, which is a known target of the NF-kappaB pathway. Collectively, the above results suggest that HHV8 vFLIP-induced NF-kappaB activation may contribute to cellular transformation seen in association with HHV8 infection by preventing the apoptosis of cells destined to die because of growth factor deprivation.

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