Immunomodulatory drugs target IKZF1-IRF4-MYC axis in primary effusion lymphoma in a cereblon-dependent manner and display synergistic cytotoxicity with BRD4 inhibitors.

Primary effusion lymphoma (PEL) is an aggressive type of non-Hodgkin lymphoma localized predominantly in body cavities. Kaposi's sarcoma-associated herpes virus (KSHV) 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 Food and Drug Administration-approved drugs for the treatment of various ailments. IMiDs display pronounced antiproliferative 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-mediated knockdown of MYC enhanced the cytotoxicity of IMiDs. Bromodomain (BRD) and extra-terminal 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 the 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 with either agent alone. These results provide compelling evidence for clinical testing of IMiDs alone and in combination with BRD4 inhibitors for PEL.

Targeting Myc in KSHV-associated primary effusion lymphoma with BET bromodomain inhibitors.

Primary effusion lymphoma (PEL) is an aggressive form of non-Hodgkin's B-cell lymphoma associated with infection by Kaposi's sarcoma-associated herpes virus (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 overexpressed 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 bromodomain and extra-terminal (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 overexpressed.

Kaposi's sarcoma-associated herpesvirus-encoded viral FLICE inhibitory protein (vFLIP) K13 suppresses CXCR4 expression by upregulating miR-146a.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV)-encoded viral FLICE inhibitory protein (vFLIP) K13 is a potent activator of the nuclear factor-kappaB (NF-kappaB) pathway. In this study, we show that infection with KHSV and ectopic expression of K13, but not its NF-kappaB-defective mutant, suppressed the expression of CXCR4. Suppression of CXCR4 by KSHV and K13 was associated with upregulated expression of miR-146a, a microRNA that is known to bind to the 3'-untranslated region of CXCR4 mRNA. Reporter studies identified two NF-kappaB sites in the promoter of miR-146a that were essential for its activation by K13. Accordingly, ectopic expression of K13, but not its NF-kappaB-defective mutant or other vFLIPs, strongly stimulated the miR-146a promoter activity, which could be blocked by specific genetic and pharmacological inhibitors of the NF-kappaB pathway. Finally, expression of CXCR4 was downregulated in clinical samples of KS and this was accompanied by an increased expression of miR-146a. Our results show that K13-induced NF-kappaB activity suppresses CXCR4 through upregulation of miR-146a. Downregulation of CXCR4 expression by K13 may contribute to KS development by promoting premature release of KSHV-infected endothelial progenitors into the circulation.

A nuclear role for Kaposi's sarcoma-associated herpesvirus-encoded K13 protein in gene regulation.

Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded viral FLICE inhibitory protein K13 interacts with a cytosolic IkappaB kinase (IKK) complex to activate nuclear factor-kappaB (NF-kappaB). We recently reported that K13 antagonizes KSHV lytic regulator RTA (replication and transcription activator) and blocks lytic replication, but spares RTA-induced viral interleukin-6 (vIL6). Here we report that K13 is also present in the nuclear compartment, a property not shared by its structural homologs. K13 interacts with and activates the nuclear IKK complex, and binds to the IkappaBalpha promoter. K13 mutants that are retained in the cytosol lack NF-kappaB activity. However, neither the IKKs nor NF-kappaB activation is required for nuclear localization of K13. Instead, this ability is dependent on a nuclear localization signal located in its N-terminal 40 amino acids. Finally, K13, along with p65/RelA, binds to the promoters of a number of KSHV lytic genes, including RTA, ORF57 and vGPCR, but not to the promoter of the vIL6 gene. Thus, K13 has an unexpected nuclear role in viral and cellular gene regulation and its differential binding to the promoters of lytic genes may not only contribute to the inhibition of KSHV lytic replication, but may also account for the escape of vIL6 from K13-induced transcriptional suppression.

Adenoviral-mediated gene transfer of ectodysplasin-A2 results in induction of apoptosis and cell-cycle arrest in osteosarcoma cell lines.

The extremely poor prognosis of patients with metastatic osteosarcoma indicates the need for novel therapeutic approaches. Ectodysplasin-A2 (EDA-A2) is a recently isolated member of the tumor necrosis factor superfamily that binds to X-linked ectodermal dysplasia receptor (XEDAR). In this report, we have analyzed the biological activity of EDA-A2 against osteosarcoma-derived cell lines. We report that XEDAR is expressed in cell lines derived from osteosarcoma and adenoviral-mediated expression of EDA-A2 in these cells results in the induction of apoptosis via caspase activation and cell-cycle arrest in the G(0)/G(1) phase. Treatment with EDA-A2 also upregulates the expression of alkaline phosphatase, a marker of osteogenic differentiation, in a caspase-dependent fashion. Collectively, our results suggest that EDA-A2 may be a promising agent for the gene therapy of osteosarcoma.

Induction of spindle cell morphology in human vascular endothelial cells by human herpesvirus 8-encoded viral FLICE inhibitory protein K13.

Human herpesvirus 8 (HHV8), also known as Kaposi's sarcoma-associated herpesvirus, is linked to the development of Kaposi's sarcoma, a disease characterized by the presence of distinctive proliferating spindle-like cells. Although HHV8 can induce spindle cell transformation of vascular endothelial cells in vitro, the viral gene(s) responsible for this phenotype remain to be identified. We demonstrate that expression of HHV8-encoded viral Fas-associated death domain protein-like IL-1beta-converting enzyme inhibitory protein K13 is sufficient to induce spindle cell phenotype in human umbilical vein endothelial cells (HUVEC), which is associated with the activation of the nuclear factor-kappaB (NF-kappaB) pathway and can be blocked by Bay-11-7082, a specific inhibitor of this pathway. K13 induces the expression of several genes known to be upregulated in HHV8-transformed vascular endothelial cells, such as interleukin (IL)-6, IL-8, CXC ligand 3 (CXCL3), orphan G protein coupled receptor (RDC1), cyclooxygenase-2 (COX-2) and dual-specificity phosphatase 5 (DUSP5). Furthermore, similar to K13, HHV8-induced spindle cell transformation of HUVEC is associated with NF-kappaB activation and can be blocked by Bay-11-7082. Thus, ectopic expression of a single latent gene of HHV8 is sufficient for the acquisition of spindle cell phenotype by vascular endothelial cells and NF-kappaB activation plays an essential role in this process.

Induction of IL-8 expression by human herpesvirus 8 encoded vFLIP K13 via NF-kappaB activation.

Human herpesvirus 8 (HHV-8) encodes a viral FLICE inhibitory protein (vFLIP), called K13, with homology to the prodomain of caspase 8. K13 has been postulated to protect virally infected cells against death receptor-induced apoptosis. We report that K13 leads to constitutive upregulation of IL-8 secretion by transcriptional upregulation of its promoter. K13-induced IL-8 promoter activation is dependent on an intact NF-kappaB-binding site and is associated with increased binding of classical NF-kappaB pathway subunits p65, c-Rel and p50, respectively. IL-8 production is defective in K13 mutants defective in classical NF-kappaB activation and is blocked by genetic and pharmacological inhibitors of this pathway. In contrast, K13 failed to activate the JNK/AP-1 pathway and deletion of AP-1-binding site in the IL-8 promoter or use of a specific JNK inhibitor had only a partial effect on K13-induced IL-8 promoter activation. Collectively, above results demonstrate that K13 is a major mediator of IL-8 production and therapeutic agents targeting K13-induced NF-kappaB pathway may have a role in the treatment of conditions in which HHV-8-induced IL-8 production plays a pathogenic role.

Inhibition of the death receptor pathway by cFLIP confers partial engraftment of MHC class I-deficient stem cells and reduces tumor clearance in perforin-deficient mice.

NK cells mediate acute rejection of MHC class I-deficient bone marrow cell (BMC) grafts. However, the exact cytotoxic mechanisms of NK cells during acute BMC graft rejection are not well defined. Although the granule exocytosis pathway plays a major role in NK cell-mediated rejection, alternative perforin-independent mechanisms also exist. By analyzing the anti-apoptotic effects of cellular Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein (cFLIP) overexpression, we investigated the possible role of death receptor-induced apoptosis in NK cell-mediated cytotoxicity. In the absence of perforin, we found that cFLIP overexpression reduces lysis of tumor cells by NK cells in vitro and in vivo. In addition, perforin-deficient NK cells were impaired in their ability to acutely reject cFLIP-overexpressing TAP-1 knockout stem cells. These results emphasize the importance of NK cell death receptor-mediated killing during BMC grafts in the absence of perforin.

Cytotoxicity of Tumor necrosis factor related apoptosis-inducing ligand towards Ewing's sarcoma cell lines.

Death ligands of the Tumor Necrosis Factor (TNF) family are known to induce apoptosis upon binding to their cognate receptors. However, the clinical utility of these cytokines as anticancer agents has been limited due to unacceptable toxicity. TRAIL is a recently isolated death ligand that possesses selective anti-tumor activity against a number of cancer cell lines without significant systemic toxicity. In this report we present evidence that cell lines derived from Ewing's Sarcoma (ES) are uniformly sensitive to TRAIL-mediated apoptosis. Furthermore, unlike TNF-alpha, treatment with TRAIL fails to induce the anti-apoptotic and pro-inflammatory NF-kappaB pathway in the ES cell lines. Our results suggest that TRAIL may prove to be a useful agent for the treatment of Ewing's sarcoma and related peripheral neuroectodermal tumors.

The ectodermal dysplasia receptor activates the nuclear factor-kappaB, JNK, and cell death pathways and binds to ectodysplasin A.

The ectodermal dysplasia receptor (EDAR) is a recently isolated member of the tumor necrosis factor receptor family that has been shown to play a key role in the process of ectodermal differentiation. We present evidence that EDAR is capable of activating the nuclear factor-kappaB, JNK, and caspase-independent cell death pathways and that these activities are impaired in mutants lacking its death domain or those associated with anhidrotic ectodermal dysplasia and the downless phenotype. Although EDAR possesses a death domain, it did not interact with the death domain-containing adaptor proteins TRADD and FADD. EDAR successfully interacted with various TRAF family members; however, a dominant-negative mutant of TRAF2 was incapable of blocking EDAR-induced nuclear factor-kappaB or JNK activation. Collectively, the above results suggest that EDAR utilizes a novel signal transduction pathway. Finally, ectodysplasin A can physically interact with the extracellular domain of EDAR and thus represents its biological ligand.