Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), or human herpesvirus 8, is a lymphotropic virus strongly linked to several AIDS-related neoplasms. The primary reservoir of infection consists of latently infected B lymphocytes and possibly other mononuclear cells. Viral reactivation from latency and spread from this lymphoid reservoir is presumably required for development of nonlymphoid tumors like KS. Here we show that deregulated expression of a single viral gene, ORF 50, which encodes a transactivator able to selectively upregulate delayed-early viral genes, suffices to disrupt latency and induce the lytic gene cascade in latently infected B cells. The identification of this gene opens the way to studies of the physiologic mechanisms controlling reactvation of KSHV from latency.

In this study we examine the molecular basis for the synergistic regulation of the minimal TCRa enhancer by multiple proteins. We find that reconstitution of TCRc~ enhancer function in nonlymphoid cells requires expression of the lymphoid-specific proteins LEF-1, Ets-1 and PEBP2a (CBFc~I, and a specific arrangement of their binding sites in the enhancer. We show that Ets-1 cooperates with PEBP2a to bind adjacent sites at one end of the enhancer, forming a ternary complex that is unstable by itself. Stable occupancy of the Ets-1-and PEBP2oL-binding sites in a DNase I protection assay was found to depend on both a specific helical phasing relationship with a nonadjacent ATF/CREB-binding site at the other end of the enhancer and on LEF-1. The HMG domain of LEF-1 was found previously to bend the DNA helix in the center of the TCRot enhancer. We now show that the HMG domain of the distantly related SRY protein, which also bends DNA, can partially replace LEF-1 in stimulating enhancer function in transfection assays. Taken together with the observation that Ets-1 and members of the ATF/CREB family have the potential to associate in vitro, these data suggest that LEF-1 can coordinate the assembly of a specific higher-order enhancer complex by facilitating interactions between proteins bound at nonadjacent sites.

Kaposi's sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus-8) establishes latent and lytic infections in both lymphoid and endothelial cells and has been associated with diseases of both cell types. The KSHV open reading frame 50 (ORF50) protein is a transcriptional activator that plays a central role in the reactivation of lytic viral replication from latency. Here we identify and characterize a DNA binding site for the ORF50 protein that is shared by the promoters of two delayed early genes (ORF57 and K-bZIP). Transfer of this element to heterologous promoters confers on them high-level responsiveness to ORF50, indicating that the element is both necessary and sufficient for activation. The element consists of a conserved 12-bp palindromic sequence and less conserved sequences immediately 3 to it. Mutational analysis reveals that sequences within the palindrome are critical for binding and activation by ORF50, but the presence of a palindrome itself is not absolutely required. The 3 flanking sequences also play a critical role in DNA binding and transactivation. The strong concordance of DNA binding in vitro with transcriptional activation in vivo strongly implies that sequence-specific DNA binding is necessary for ORF50-mediated activation through this element. Expression of truncated versions of the ORF50 protein reveals that DNA binding is mediated by the amino-terminal 272 amino acids of the polypeptide.Infection by Kaposi's sarcoma-associated herpesvirus (KSHV) also known as human herpesvirus 8 (HHV-8) is associated with malignancies of both endothelial and lymphoid cells in humans. KSHV has been well established as the etiologic agent responsible for Kaposi's sarcoma (KS), an endothelial neoplasm frequent in homosexual men with AIDS and highly prevalent in sub-Saharan Africa (reviewed in reference 25). KSHV is also linked to two other AIDS-related malignancies, primary effusion lymphoma and multicentric Castleman's disease (5, 26). The presence of viral DNA in CD19 ϩ B cells and other mononuclear cells of the peripheral blood of KS/ AIDS patients (1, 2, 31), even prior to full-blown KS, suggests that infection of the lymphoid compartment is antecedent to the development of the endothelial disease.KSHV infection, similar to infection by other herpesviruses, displays two life cycle modes, latency and lytic replication. Latency is established by the virus both in endothelial and B cells and is detectable in such cell types both in vitro and in infected hosts (1,3,7,19,22,27,31). KSHV latency-associated genes are expressed in most spindle cells of KS tumors and are thought to contribute to their survival and proliferation (25). However, many viral genes (e.g., vGCR and vMIPs I and II) encoding homologs of cellular signaling proteins which have been suspected of roles in the histogenesis of KS are expressed strictly as lytic cycle products (13,20,23,24,28). This suggests that the KSHV lytic cycle may also contribute to KS lesion formation. Additional support for this notion comes from stud...

Elesclomol (formerly STA-4783) is a novel small molecule undergoing clinical evaluation in a pivotal phase III melanoma trial (SYMMETRY). In a phase II randomized, double-blinded, controlled, multi-center trial in 81 patients with stage IV metastatic melanoma, treatment with elesclomol plus paclitaxel showed a statistically significant doubling of progression-free survival time compared with treatment with paclitaxel alone. Although elesclomol displays significant therapeutic activity in the clinic, the mechanism underlying its anticancer activity has not been defined previously. Here, we show that elesclomol induces apoptosis in cancer cells through the induction of oxidative stress. Treatment of cancer cells in vitro with elesclomol resulted in the rapid generation of reactive oxygen species (ROS) and the induction of a transcriptional gene profile characteristic of an oxidative stress response. Inhibition of oxidative stress by the antioxidant N-acetylcysteine blocked the induction of gene transcription by elesclomol. In addition, N-acetylcysteine blocked drug-induced apoptosis, indicating that ROS generation is the primary mechanism responsible for the proapoptotic activity of elesclomol. Excessive ROS production and elevated levels of oxidative stress are critical biochemical alterations that contribute to cancer cell growth. Thus, the induction of oxidative stress by elesclomol exploits this unique characteristic of cancer cells by increasing ROS levels beyond a threshold that triggers cell death.

Open reading frame (ORF) 57 of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a homolog of known posttranscriptional regulators that are essential for replication in other herpesviruses. Here, we examined the expression of this gene and the function(s) of its product. KSHV ORF 57 is expressed very early in infection from a 1.6-kb spliced RNA bearing several in-frame initiation codons. Its product is a nuclear protein that, in transient assays, has little effect on the expression of luciferase reporter genes driven by a variety of KSHV and heterologous promoters. However, ORF 57 protein enhances the accumulation of several viral transcripts, in a manner suggesting posttranscriptional regulation. These transcripts include not only known cytoplasmic mRNAs (e.g., ORF 59) but also a nuclear RNA (nut-1) that lacks coding potential. Finally, ORF 57 protein can also modulate the effects of the ORF 50 gene product, a classical transactivator known to be required for lytic induction. The expression from some (e.g., nut-1) but not all (e.g., tk) ORF 50-responsive promoters can be synergistically enhanced by coexpression of ORF 50 and ORF 57. This effect is not due to upregulation of ORF 50 expression but rather to a posttranslational enhancement of the transcriptional activity of ORF 50. These data indicate that ORF 57 is a powerful pleiotropic effector that can act on several posttranscriptional levels to modulate the expression of viral genes in infected cells.Kaposi's sarcoma (KS), an endothelial tumor with neoangiogenic and inflammatory components, is a common neoplasm of AIDS patients. Recent evidence strongly implicates a novel lymphotropic herpesvirus, KS-associated herpesvirus (KSHV; also called human herpesvirus 8), in the pathogenesis of KS (5, 12; for reviews, see references 14 and 41). KSHV DNA is found in virtually all of the spindle (endothelial) cells of clinically apparent KS lesions (8, 46), as well as in tumorinfiltrating monocytes (6) and circulating B cells (1,27,50). Consistent with its classification as a lymphotropic gammaherpesvirus, KSHV is also tightly linked to certain B-cell lymphomas, termed primary effusion lymphomas (9, 45). Although KSHV infection of KS spindle cells is predominantly latent, lytic replication is also evident in the tumor (28,35,36,46), and growing evidence suggests that the lytic cycle contributes importantly to tumorigenesis. For example, the incidence of KS is greatly decreased when AIDS patients at risk for KS are treated with ganciclovir, a drug that specifically blocks lytic viral replication (25). Moreover, several lytic-cycle gene products can stimulate inflammatory and angiogenic responses in surrounding cells and tissues (2, 3, 7). Lytic replication has also been posited to be required for KSHV spread from its presumed lymphoreticular reservoir to its endothelial targets (24). Thus, the study of the KSHV lytic cycle (and the switch from latency to lytic growth) is important not only to fully characterize the molecular basis of viral replication but also to f...

Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV) is a lymphotropic virus strongly linked to the development of KS, an endothelial cell neoplasm frequent in persons with AIDS. Reactivation from latency in B cells is thought to be an important antecedent to viral spread to endothelial cells during KS pathogenesis. Earlier experiments have posited a role for the transcriptional activator encoded by KSHV open reading frame 50 (ORF50) in such reactivation, since ectopic overexpression of this protein induces reactivation in latently infected B cells. Here we have explored several aspects of the expression, structure, and function of this protein bearing on this role. The ORF50 gene is expressed very early in lytic reactivation, before several other genes implicated as candidate regulatory genes in related viruses, and its expression can upregulate their promoters in transient assays. The protein is extensively phosphorylated in vivo and bears numerous sites for phosphorylation by protein kinase C, activators of which are potent stimulators of lytic induction. The C terminus of the ORF50 protein contains a domain that can strongly activate transcription when targeted to DNA; deletion of this domain generates an allele that expresses a truncated protein which retains the ability to form multimers with full-length ORF50 and functions as a dominant-negative protein. Expression of this allele in latently infected cells ablates spontaneous reactivation from latency and strikingly suppresses viral replication induced by multiple stimuli, including phorbol ester, ionomycin, and sodium butyrate. These results indicate that the ORF50 gene product plays an essential role in KSHV lytic replication and are consistent with its action as a putative molecular switch controlling the induction of virus from latency.

Bispecific antibodies, while showing great therapeutic potential, pose formidable challenges with respect to their assembly, stability, immunogenicity, and pharmacodynamics. Here we describe a novel class of bispecific antibodies with native human immunoglobulin format. The design exploits differences in the affinities of the immunoglobulin isotypes for Protein A, allowing efficient large-scale purification. Using this format, we generated a bispecific antibody, REGN1979, targeting the B cell marker, CD20, and the CD3 component of the T cell receptor, which triggers redirected killing of B cells. In mice, this antibody prevented growth of B cell tumors and also caused regression of large established tumors. In cynomolgus monkeys, low doses of REGN1979 caused prolonged depletion of B cells in peripheral blood with a serum half-life of approximately 14 days. Further, the antibody induced a deeper depletion of B cells in lymphoid organs than rituximab. This format has broad applicability for development of clinical bispecific antibodies.

T cell activation is initiated upon binding of the T cell receptor (TCR)/CD3 complex to peptide–major histocompatibility complexes (“signal 1”); activation is enhanced by engagement of a second “costimulatory” receptor, such as the CD28 receptor on T cells binding to its cognate ligand(s) on the target cell (“signal 2”). CD3-based bispecific antibodies act by replacing conventional signal 1, linking T cells to tumor cells by binding a tumor-specific antigen (TSA) with one arm of the bispecific and bridging to TCR/CD3 with the other. Although some of these so-called TSAxCD3 bispecifics have demonstrated promising antitumor efficacy in patients with cancer, their activity remains to be optimized. Here, we introduce a class of bispecific antibodies that mimic signal 2 by bridging TSA to the costimulatory CD28 receptor on T cells. We term these TSAxCD28 bispecifics and describe two such bispecific antibodies: one specific for ovarian and the other for prostate cancer antigens. Unlike CD28 superagonists, which broadly activate T cells and resulted in profound toxicity in early clinical trials, these TSAxCD28 bispecifics show limited activity and no toxicity when used alone in genetically humanized immunocompetent mouse models or in primates. However, when combined with TSAxCD3 bispecifics, they enhance the artificial synapse between a T cell and its target cell, potentiate T cell activation, and markedly improve antitumor activity of CD3 bispecifics in a variety of xenogeneic and syngeneic tumor models. Combining this class of CD28-costimulatory bispecific antibodies with the emerging class of TSAxCD3 bispecifics may provide well-tolerated, off-the-shelf antibody therapies with robust antitumor efficacy.