BC-3 Cells Research Articles

Arsenic Trioxide and Lenalidomide: A Promising Combination Therapy for Primary Effusion Lymphoma

Primary Effusion Lymphoma (PEL) is an aggressive malignancy caused by the Kaposi sarcoma associated herpesvirus (KSHV). PEL patients rarely respond to conventional chemotherapy and their prognosis is poor, with a median survival of less than six months. The most promising treatment strategies in these patients combine anti-viral treatments with new agents including chemicals and cytokines. Here, we investigated the efficacy of lenalidomide (Lena), an immune modulator with significant clinical activity in a large number of hematological malignancies, and arsenic trioxide (As), a potent agent in acute promyelocytic leukemia and HTLV-I associated adult T cell leukemia.We used three PEL cell lines (BC-1, BC-3 and BCBL-1 which are KSHV+/EBV- malignant B cells derived from PEL patients) and one PEL-negative control cell line (RAJI as a KSHV- malignant B cell line). We demonstrate that the combination of As and Lena resulted in growth inhibition in BC-1 and BC-3 cells and to a lesser degree in BCBL-1 cells while no effect was observed in RAJI cells. This growth arrest was associated with induction of massive apoptosis and a substantial upregulation of phosphorylated and total p53 protein levels. Furthermore, As/Lena inhibited the expression of latent viral transcripts (LANA-1, LANA-2, v-FLIP and v-Cyclin) and proteins (LANA-1/LANA-2) in all tested PEL cells.Finally, and while Lena alone did not have any inhibitory effect on in vitro treated PEL cells, single agent Lena treatment doubled survival of BC-3 PEL mice and significantly prolonged survival in 75% of BCBL-1 PEL mice and led to cure of the remaining 25%. More importantly, combination of As and Lena was highly synergistic and cured 25% and 75% of BC-3 and BCBL-1 PEL mice respectively. These results provide a rational for the therapeutic use of the combination As/Lena in PEL patients. DisclosuresNo relevant conflicts of interest to declare.

Kaposi's Sarcoma-Associated Herpesvirus-Encoded circRNAs Are Expressed in Infected Tumor Tissues and Are Incorporated into Virions.

Kaposi's sarcoma-associated herpesvirus (KSHV) has recently been found to generate circular RNAs (circRNAs) from several KSHV genes, most abundantly from K10 (viral interferon regulatory factor 4 [vIRF4]), K7.3, and polyadenylated nuclear (PAN) RNA. To define expression of these circRNAs, KSHV-infected cell lines, patient tissues, and purified virions were examined. KSHV circRNA expression was universally detected in tests of six primary effusion lymphoma (PEL) cell lines but ranged from low-level expression in BC-1 cells dually infected with tightly latent KSHV and Epstein-Barr virus to abundant expression in KSHV-only BCBL-1 cells with spontaneous virus production. Generally, the PAN/K7.3 locus broadly and bidirectionally generated circRNA levels that paralleled the corresponding linear RNA levels. However, RNA corresponding to a particular KSHV circularization site (circ-vIRF4) was minimally induced, despite linear vIRF4 RNA being activated by virus induction. In situ hybridization showed abundant circ-vIRF4 in noninduced PEL cells. All three KSHV circRNAs were isolated as nuclease-protected forms from gradient-purified virions collected from BrK.219 cells infected with a KSHV molecular clone. For circ-vIRF4, the fully processed form that is exported to the cytoplasm was incorporated into virus particles but the nuclear, intron-retaining form was not. The half-life of circ-vIRF4 was twice as long as that of its linear counterpart. The KSHV circRNAs could be detected at a higher rate than their corresponding linear counterparts by in situ hybridization in archival tissues and by reverse transcription-PCR (RT-PCR) in sera stored for over 25 years. In summary, KSHV circRNAs are expressed in infection-associated diseases, can be regulated depending on virus life cycle, and are incorporated into viral particles for preformed delivery, suggesting a potential function in early infection.IMPORTANCE KSHV has recently been found to encode circRNAs. circRNAs result from back-splicing of an upstream pre-mRNA splice donor exon-intron junction to an acceptor site, generating a covalently closed circle. This study revealed that for one KSHV region, the PAN/K7.3 locus, broadly and bidirectionally generated circRNA levels parallel corresponding linear RNA levels. Another KSHV circularization site (circ-vIRF4), however, showed expression that differed from that of the corresponding linear RNA. All KSHV circRNAs are incorporated into KSHV virions and are potentially expressed as immediate early products in newly infected cells.

Reduction of Kaposi's Sarcoma-Associated Herpesvirus Latency Using CRISPR-Cas9 To Edit the Latency-Associated Nuclear Antigen Gene.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS), an AIDS-defining cancer in HIV-1-infected individuals or immune-suppressed transplant patients. The prevalence for both KSHV and KS are highest in sub-Saharan Africa where HIV-1 infection is also epidemic. There is no effective treatment for advanced KS; therefore, the survival rate is low. Similar to other herpesviruses, KSHV's ability to establish latent infection in the host presents a major challenge to KS treatment or prevention. Strategies to reduce KSHV episomal persistence in latently infected cells might lead to approaches to prevent KS development. The CRISPR-Cas9 system is a gene editing technique that has been used to specifically manipulate the HIV-1 genome but also Epstein-Barr virus (EBV) which, similar to KSHV, belongs to the Gammaherpesvirus family. Among KSHV gene products, the latency-associated nuclear antigen (LANA) is absolutely required in the maintenance, replication, and segregation of KSHV episomes during mitosis, which makes LANA an ideal target for CRISPR-Cas9 editing. In this study, we designed a replication-incompetent adenovirus type 5 to deliver a LANA-specific Cas9 system (Ad-CC9-LANA) into various KSHV latent target cells. We showed that KSHV latently infected epithelial and endothelial cells transduced with Ad-CC9-LANA underwent significant reductions in the KSHV episome burden, LANA RNA and protein expression over time, but this effect is less profound in BC3 cells due to the low infection efficiency of adenovirus type 5 for B cells. The use of an adenovirus vector might confer potential in vivo applications of LANA-specific Cas9 against KSHV infection and KS.IMPORTANCE The ability for Kaposi's sarcoma-associated herpesvirus (KSHV), the causative agent of Kaposi's sarcoma (KS), to establish and maintain latency has been a major challenge to clearing infection and preventing KS development. This is the first study to demonstrate the feasibility of using a KSHV LANA-targeted CRISPR-Cas9 and adenoviral delivery system to disrupt KSHV latency in infected epithelial and endothelial cell lines. Our system significantly reduced the KSHV episomal burden over time. Given the safety record of adenovirus as vaccine or delivery vectors, this approach to limit KSHV latency may also represent a viable strategy against other tumorigenic viruses and may have potential benefits in developing countries where the viral cancer burden is high.

Importance of Promyelocytic Leukema Protein (PML) for Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication.

Many DNA virus replication-related proteins are associated with promyelocytic leukemia protein (PML), a component of nuclear domain 10 (ND10), which has been investigated for its potential involvement in viral replication. In the case of Kaposi’s sarcoma-associated herpesvirus (KSHV) lytic gene products, K8 (K-bZIP), ORF59, and ORF75 have been shown to colocalize with PML, but its importance in KSHV lytic replication is still unclear. In this study, we analyzed the functional influence of PML on KSHV latency and lytic replication in KSHV-infected primary effusion lymphoma (PEL) cell lines. Stable PML-knockout (BC3-PMLKO) and PML-overexpressing BC3 cells (BC3PML) were successfully generated and the latency and reactivation status were analyzed. The results demonstrated that neither KSHV latency nor the episome copy number was affected in BC3-PMLKO cells. In the reactivation phase, the expression dynamics of KSHV immediate-early or early lytic proteins such as RTA, K9 (vIRF1), K5, K3, ORF59, and K8 (K-bZIP) were comparable between wild-type, control BC3, and BC3-PMLKO cells. Interestingly, KSHV lytic replication, virion production, and expression of late genes were downregulated in BC3-PMLKO cells and upregulated in BC3PML cells, compared to those in control or wild-type BC3 cells. Moreover, exogenous PML increased the size of the PML dots and recruited additional K8 (K-bZIP) to PML-NBs as dots. Therefore, PML would function as a positive regulator for KSHV lytic DNA replication by recruiting KSHV replication factors such as 8 (K-bZIP) or ORF59 to the PML-NBs.

IRE1\u03b1 deficiency promotes tumor cell death and eIF2\u03b1 degradation through PERK dipendent autophagy

Sensors of endoplasmic reticulum (ER) stress function in a co-ordinated manner. In the present study we investigated the relationship between IRE1α and PERK pathways and survival of ER stressed U937 cells and BC3 cells. To this end, we investigated the effects of a subcytotoxic concentration of Tunicamycin in IRE1α-proficient and in IRE1α-deficient cells, by pharmacological inhibition with 4μ8 C or down-regulation by specific siRNA. We show that either type of IRE1α deficiency affects eIF2α expression and causes cell death increase. GSK2606414, a PERK inhibitor, and PERK specific siRNA prevent eIF2α down-regulation and restore cell survival. Degradation of this protein is due to autophagy, as it is prevented by bafilomycin and not by proteasome inhibition. Furthermore, activation of the autophagy flux is PERK dependent. Also the Cathepsin B inhibitor CA074 prevents eIF2α from degradation and reduces cell death. Altogether, these results show that IRE1α deficiency in ER stressed cells leads to an unexpected decrease of eIF2α, an important molecule for protein translation, through PERK dependent autophagy. Thus, IRE1/XBP1 inhibitors may represent a feasible strategy for tumor therapy, while PERK inhibitors may vanish the goal.

Nuclear Innate Immune DNA Sensor IFI16 Is Degraded during Lytic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus (KSHV): Role of IFI16 in Maintenance of KSHV Latency.

IFI16 (interferon gamma-inducible protein 16) recognizes nuclear episomal herpesvirus (Kaposi's sarcoma-associated herpesvirus [KSHV], Epstein-Barr virus [EBV], and herpes simplex virus 1 [HSV-1]) genomes and induces the inflammasome and interferon beta responses. It also acts as a lytic replication restriction factor and inhibits viral DNA replication (human cytomegalovirus [HCMV] and human papillomavirus [HPV]) and transcription (HSV-1, HCMV, and HPV) through epigenetic modifications of the viral genomes. To date, the role of IFI16 in the biology of latent viruses is not known. Here, we demonstrate that knockdown of IFI16 in the latently KSHV-infected B-lymphoma BCBL-1 and BC-3 cell lines results in lytic reactivation and increases in levels of KSHV lytic transcripts, proteins, and viral genome replication. Similar results were also observed during KSHV lytic cycle induction in TREX-BCBL-1 cells with the doxycycline-inducible lytic cycle switch replication and transcription activator (RTA) gene. Overexpression of IFI16 reduced lytic gene induction by the chemical agent 12-O-tetradecoylphorbol-13-acetate (TPA). IFI16 protein levels were significantly reduced or absent in TPA- or doxycycline-induced cells expressing lytic KSHV proteins. IFI16 is polyubiquitinated and degraded via the proteasomal pathway. The degradation of IFI16 was absent in phosphonoacetic acid-treated cells, which blocks KSHV DNA replication and, consequently, late lytic gene expression. Chromatin immunoprecipitation assays of BCBL-1 and BC-3 cells demonstrated that IFI16 binds to KSHV gene promoters. Uninfected epithelial SLK and osteosarcoma U2OS cells transfected with KSHV luciferase promoter constructs confirmed that IFI16 functions as a transcriptional repressor. These results reveal that KSHV utilizes the innate immune nuclear DNA sensor IFI16 to maintain its latency and repression of lytic transcripts, and a late lytic KSHV gene product(s) targets IFI16 for degradation during lytic reactivation. Like all herpesviruses, latency is an integral part of the life cycle of Kaposi's sarcoma-associated herpesvirus (KSHV), an etiological agent for many human cancers. Herpesviruses utilize viral and host factors to successfully evade the host immune system to maintain latency. Reactivation is a complex event where the latent episomal viral genome springs back to active transcription of lytic cycle genes. Our studies reveal that KSHV has evolved to utilize the innate immune sensor IFI16 to keep lytic cycle transcription in dormancy. We demonstrate that IFI16 binds to the lytic gene promoter, acts as a transcriptional repressor, and thereby helps to maintain latency. We also discovered that during the late stage of lytic replication, KSHV selectively degrades IFI16, thus relieving transcriptional repression. This is the first report to demonstrate the role of IFI16 in latency maintenance of a herpesvirus, and further understanding will lead to the development of strategies to eliminate latent infection.

Cellular MicroRNA Let-7a Suppresses KSHV Replication through Targeting MAP4K4 Signaling Pathways

BackgroundKaposi’s sarcoma (KS)-associated herpesvirus (KSHV) is the etiologic agent of KS, the most common AIDS-related malignancy. The majority of KS tumor cells harbor latent KSHV virus but only a small percentage undergoes spontaneous lytic replication. Viral reactivation from latency is crucial for the pathogenesis and development of KS, but the cellular mechanisms underlying the switch between viral latency and replication are not well understood.MethodsThe level of let-7 miRNAs and MAP4K4 in KSHV infected 293T cells were quantified by real-time PCRs. Let-7 expression was silenced by the miRNA sponge technique. In let-7a transfected 293T cells, the expression of MAP4K4 was measured by real-time PCR and western blot. Luciferease expression was employed to examine the effect of let-7a on the 3’-untranslated region (UTR) of the MAP4K4 gene in 293T cells. Real-time PCR was used to quantify the KSHV copy numbers in BC-3 cells in which the expression of let-7a and/or MAP4K4 were altered. Finally, ERK, JNK and p38 protein production and their phosphorylation status were detected by western blots in let-7a or MAP4K4 transfected BCBL-1 cells.ResultsThe expression of microRNA let-7 was dramatically decreased in KSHV infected 293T cells, but that of MAP4K4 was increased significantly. Let-7a is physically associated with and targets the MAP4K4 3’UTR, and inhibits MAP4K4 expression at both mRNA and protein levels. MAP4K4 stimulates KSHV reactivation from latency, whereas let-7a inhibits the function of MAP4K4 by reversing the function of MAP4K4 on JNK, phospho-JNK and phospho-ERK1/2 levels.ConclusionOur results establish that let-7a specifically suppresses MAP4K4 expression, and further inhibits KSHV reactivation by interfering with the function of MAP4K4 on the MAPK pathway, highlighting let-7a as a potential treatment for KS.

A Screen for Extracellular Signal-Regulated Kinase-Primed Glycogen Synthase Kinase 3 Substrates Identifies the p53 Inhibitor iASPP.

The Kaposi's sarcoma-associated herpesvirus (KSHV) LANA protein is essential for the replication and maintenance of virus genomes in latently KSHV-infected cells. LANA also drives dysregulated cell growth through a multiplicity of mechanisms that include altering the activity of the cellular kinases extracellular signal-regulated kinase (ERK) and glycogen synthase kinase 3 (GSK-3). To investigate the potential impact of these changes in enzyme activity, we used protein microarrays to identify cell proteins that were phosphorylated by the combination of ERK and GSK-3. The assays identified 58 potential ERK-primed GSK-3 substrates, of which 23 had evidence for in vivo phosphorylation in mass spectrometry databases. Two of these, SMAD4 and iASPP, were selected for further analysis and were confirmed as ERK-primed GSK-3 substrates. Cotransfection experiments revealed that iASPP, but not SMAD4, was targeted for degradation in the presence of GSK-3. iASPP interferes with apoptosis induced by p53 family members. To determine the importance of iASPP to KSHV-infected-cell growth, primary effusion lymphoma (PEL) cells were treated with an iASPP inhibitor in the presence or absence of the MDM2 inhibitor Nutlin-3. Drug inhibition of iASPP activity induced apoptosis in BC3 and BCBL1 PEL cells but did not induce poly(ADP-ribose) polymerase (PARP) cleavage in virus-negative BJAB cells. The effect of iASPP inhibition was additive with that of Nutlin-3. Interfering with iASPP function is therefore another mechanism that can sensitize KSHV-positive PEL cells to cell death. KSHV is associated with several malignancies, including primary effusion lymphoma (PEL). The KSHV-encoded LANA protein is multifunctional and promotes both cell growth and resistance to cell death. LANA is known to activate ERK and limit the activity of another kinase, GSK-3. To discover ways in which LANA manipulation of these two kinases might impact PEL cell survival, we screened a human protein microarray for ERK-primed GSK-3 substrates. One of the proteins identified, iASPP, showed reduced levels in the presence of GSK-3. Further, blocking iASPP activity increased cell death, particularly in p53 wild-type BC3 PEL cells.

KSHV miRNAs Decrease Expression of Lytic Genes in Latently Infected PEL and Endothelial Cells by Targeting Host Transcription Factors

Kaposi’s sarcoma-associated herpesvirus (KSHV) microRNAs are encoded in the latency-associated region. Knockdown of KSHV miR-K12-3 and miR-K12-11 increased expression of lytic genes in BC-3 cells, and increased virus production from latently infected BCBL-1 cells. Furthermore, iSLK cells infected with miR-K12-3 and miR-K12-11 deletion mutant viruses displayed increased spontaneous reactivation and were more sensitive to inducers of reactivation than cells infected with wild type KSHV. Predicted binding sites for miR-K12-3 and miR-K12-11 were found in the 3’UTRs of the cellular transcription factors MYB, Ets-1, and C/EBPα, which activate RTA, the KSHV replication and transcription activator. Targeting of MYB by miR-K12-11 was confirmed by cloning the MYB 3’UTR downstream from the luciferase reporter. Knockdown of miR‑K12-11 resulted in increased levels of MYB transcript, and knockdown of miR-K12-3 increased both C/EBPα and Ets-1 transcripts. Thus, miR-K12-11 and miR-K12-3 contribute to maintenance of latency by decreasing RTA expression indirectly, presumably via down‑regulation of MYB, C/EBPα and Ets-1, and possibly other host transcription factors.

Epidemiology and genetic variability of HHV-8/KSHV in Pygmy and Bantu populations in Cameroon.

BackgroundKaposi's sarcoma associated herpesvirus (KSHV/HHV-8) is the causal agent of all forms of Kaposi sarcoma. Molecular epidemiology of the variable K1 region identified five major subtypes exhibiting a clear geographical clustering. The present study is designed to gain new insights into the KSHV epidemiology and genetic diversity in Cameroon.Methodology/Principal FindingsBantu and Pygmy populations from remote rural villages were studied. Antibodies directed against latent nuclear antigens (LANA) were detected by indirect immunofluorescence using BC3 cells. Peripheral blood cell DNAs were subjected to a nested PCR amplifying a 737 bp K1 gene fragment. Consensus sequences were phylogenetically analyzed. We studied 2,063 persons (967 females, 1,096 males, mean age 39 years), either Bantus (1,276) or Pygmies (787). The Bantu group was older (42 versus 35 years: P<10−4). KSHV anti-LANA seroprevalence was of 37.2% (768/2063), with a significant increase with age (P<10−4) but no difference according to sex. Seroprevalence, as well as the anti-LANA antibodies titres, were higher in Bantus (43.2%) than in Pygmies (27.6%) (P<10−4), independently of age. We generated 29 K1 sequences, comprising 24 Bantus and five Pygmies. These sequences belonged to A5 (24 cases) or B (five cases) subtypes. They exhibited neither geographical nor ethnic aggregation. A5 strains showed a wide genetic diversity while the B strains were more homogenous and belonged to the B1 subgroup.ConclusionThese data demonstrate high KSHV seroprevalence in the two major populations living in Southern and Eastern Cameroon with presence of mostly genetically diverse A5 but also B K1 subtypes.

P53 Tumor Suppressor Protein Stability and Transcriptional Activity Are Targeted by Kaposi's Sarcoma-Associated Herpesvirus-Encoded Viral Interferon Regulatory Factor 3

Viruses have developed numerous strategies to counteract the host cell defense. Kaposi's sarcoma-associated herpesvirus (KSHV) is a DNA tumor virus linked to the development of Kaposi's sarcoma, Castleman's disease, and primary effusion lymphoma (PEL). The virus-encoded viral interferon regulatory factor 3 (vIRF-3) gene is a latent gene which is involved in the regulation of apoptosis, cell cycle, antiviral immunity, and tumorigenesis. vIRF-3 was shown to interact with p53 and inhibit p53-mediated apoptosis. However, the molecular mechanism underlying this phenomenon has not been established. Here, we show that vIRF-3 associates with the DNA-binding domain of p53, inhibits p53 phosphorylation on serine residues S15 and S20, and antagonizes p53 oligomerization and the DNA-binding affinity. Furthermore, vIRF-3 destabilizes p53 protein by increasing the levels of p53 polyubiquitination and targeting p53 for proteasome-mediated degradation. Consequently, vIRF-3 attenuates p53-mediated transcription of the growth-regulatory p21 gene. These effects of vIRF-3 are of biological relevance since the knockdown of vIRF-3 expression in KSHV-positive BC-3 cells, derived from PEL, leads to an increase in p53 phosphorylation, enhancement of p53 stability, and activation of p21 gene transcription. Collectively, these data suggest that KSHV evolved an efficient mechanism to downregulate p53 function and thus facilitate uncontrolled cell proliferation and tumor growth.

The Kaposi's Sarcoma-Associated Herpesvirus (KSHV)-Induced 5-Lipoxygenase-Leukotriene B4 Cascade Plays Key Roles in KSHV Latency, Monocyte Recruitment, and Lipogenesis

Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). KS lesions are characterized by endothelial cells with multiple copies of the latent KSHV episomal genome, lytic replication in a low percentage of infiltrating monocytes, and inflammatory cytokines plus growth factors. We demonstrated that KSHV utilizes inflammatory cyclooxygenase 2/prostaglandin E2 to establish and maintain latency (Sharma-Walia, N., A. G. Paul, V. Bottero, S. Sadagopan, M. V. Veettil, N. Kerur, and B. Chandran, PLoS Pathog 6:e1000777, 2010 [doi:10.1371/journal.ppat.1000777]). Here, we evaluated the role of 5-lipoxygenase (5LO) and its chemotactic metabolite leukotriene B4 (LTB4) in KSHV biology. Abundant staining of 5LO was detected in human KS tissue sections. We observed elevated levels of 5LO and high levels of secretion of LTB4 during primary KSHV infection of endothelial cells and in PEL B cells (BCBL-1 and BC-3 cells). Blocking the 5LO/LTB4 cascade inhibited viral latent ORF73, immunomodulatory K5, viral macrophage inflammatory protein 1 (MIP-1), and viral MIP-2 gene expression, without much effect on lytic switch ORF50, immediate early lytic K8, and viral interferon-regulatory factor 2 gene expression. 5LO inhibition significantly downregulated latent viral Cyclin and latency-associated nuclear antigen 2 levels in PEL cells. 5LO/LTB4 inhibition downregulated TH2-related cytokine secretion, elevated TH1-related cytokine secretion, and reduced human monocyte recruitment, adhesion, and transendothelial migration. 5LO/LTB4 inhibition reduced fatty acid synthase (FASN) promoter activity and its expression. Since FASN, a key enzyme required in lipogenesis, is important in KSHV latency, these findings collectively suggest that 5LO/LTB4 play important roles in KSHV biology and that effective inhibition of the 5LO/LTB4 pathway could potentially be used in treatment to control KS/PEL.

Inactivation Of BANK1 By a Novel IGH-Associated Translocation and 5' Hypermethylation In B-Cell Lymphomas

A novel IGH-associated reciprocal translocation, t(4;14)(q24;q32), was identified, along with trisomy 9, in 20 of 20 metaphases by conventional karyotyping in a case of malignant gastric post-transplant lymphoproliferative disorder (PTLD). Cloning of the translocation site by inverse PCR identified BANK1 (B-cell scaffold protein with ankyrin repeats 1), a B-cell-specific adaptor protein with putative functions in B-cell receptor and CD40 signaling, as a novel IGH translocation partner. The breakpoints were located at the Sα region of IGH and intron 1 of BANK1. The translocation juxtaposed the two genes in opposite orientations, and surprisingly, resulted in transcriptional inactivation of BANK1 as a result of dissociation of the major BANK1 promoter. While BANK1 isoforms were expressed in all tonsillar B-cells, with lower levels (∼ 5 fold) in the germinal centers (GC) compared to naïve and memory B-cells, transcription from the major promoter in the tumor was absent and transcription from the minor promoter was reduced 50% relative to GC B-cells, suggesting that the non-translocated BANK1 allele was also inactivated. The total BANK1 expression was very low (∼10% of normal GC B cells) and crytic promoter activation was not identified. Several genes (PPP3CA, MIR1255A, FLJ20021 and SLC39A8), located 180 to 440 kb away from BANK1, were analyzed for mRNA expression; there is no significant activation in any of these genes, further supporting that BANK1is indeed the target gene affected by the translocation.Interphase FISH using break-apart BANK1 probes confirmed breakpoint in the index case but did not identify translocations in additional 15 PTLDs and 68 diffuse large B-cell lymphomas (DLBCL), implying that BANK1 translocation may be a rare event. To determine if BANK1 inactivation may occur in B-cell lymphomas by other mechanisms, 23 B-cell lymphoma cell lines, including 8 Burkitt lymphoma (BL), 9 diffuse large B cell lymphoma (DLBCL), 3 primary effusion lymphoma (PEL), and 3 classical Hodgkin lymphoma (cHL) were bisulfite sequenced to assess the methylation status of 37 CpG dinucleotides in a 436 base-pair region at the 5' end of BANK1, which extends across exon 1 into the 5' portion of intron 1. High level of methylation (>60% methylation on average among all CpGs) was seen in all 3 cHL and 2 of 3 PEL cell lines. Regional methylation was seen in 3 of 8 BL lines and 1 of 3 PEL lines. No hypermemethylation was identified in the DLBCL lines or in normal tonsils. Hypermethylation was associated with almost complete silencing of BANK1 transcription. In the DLBCL lines and BL lines without BANK1 hypermethylation, BANK1mRNA expressions were variable, ranging from <5% to 130% of GCB cells.To confirm that BANK1 hypermethylation is present in primary lymphoma cases, methylation status of 17 of the 37 CpGs were assessed in 23 cHL cases using en bloc formalin-fixed, paraffin-embedded materials and also laser-capture micro-issected Hodgkin/Reed-Sternberg (HRS) cells. There was evidence of BANK1 hypermethylation in the tumor cells in 9 of 23 cHL. Tumor cell specificity of BANK1 hypermethylation was further confirmed in 4 cHL cases using micro-dissected HRS cells. HRS cells were negative for BANK1 in 28 of 29 cHL cases examined by immunohistochemistry, suggesting that other mechanisms other than DNA methylation may be responsible for silencing BANK1expression.To investigate whether BANK1 has biological effects on B-cells related to lymphoma development, exogenous BANK1 was re-introduced to BC3, a PEL cell line showing marked BANK1 hypermethylation with absence of BANK1 expression. We established a stable doxycycline-inducible BC3 cell line expressing BANK1. Inhibition of cell growth was observed 2 to 3 days after doxycyline induction, and the number of viable cells with transfected BANK1 was only 25% compared to BC3 cells carry vehicle alone at day 6. An analysis of 5-bromo-2' deoxyuridine (BrdU) incorporation after 48 hours of doxycline induction revealed that the fraction of cells in S-phase was reduced by 50% in the BANK1 transfectants, suggesting that BANK1has a negative effect on cell proliferation in these B cells.In summary, we have identified a novel IGH translocation partner and provide an example of an unusual consequence (gene inactivation) of IGH-associated translocation. We provide for the first time evidence of a potential role of BANK1 down-regulation in the development of B-cell lymphomas. Disclosures:No relevant conflicts of interest to declare.

Kaposi's Sarcoma-Associated Herpesvirus-Positive Primary Effusion Lymphoma Tumor Formation in NOD/SCID Mice Is Inhibited by Neomycin and Neamine Blocking Angiogenin's Nuclear Translocation

Angiogenin (ANG) is a 14-kDa multifunctional proangiogenic secreted protein whose expression level correlates with the aggressiveness of several tumors. We observed increased ANG expression and secretion in endothelial cells during de novo infection with Kaposi's sarcoma-associated herpesvirus (KSHV), in cells expressing only latency-associated nuclear antigen 1 (LANA-1) protein, and in KSHV latently infected primary effusion lymphoma (PEL) BCBL-1 and BC-3 cells. Inhibition of phospholipase Cγ (PLCγ) mediated ANG's nuclear translocation by neomycin, an aminoglycoside antibiotic (not G418-neomicin), resulted in reduced KSHV latent gene expression, increased lytic gene expression, and increased cell death of KSHV(+) PEL and endothelial cells. ANG detection in significant levels in KS and PEL lesions highlights its importance in KSHV pathogenesis. To assess the in vivo antitumor activity of neomycin and neamine (a nontoxic derivative of neomycin), BCBL-1 cells were injected intraperitoneally into NOD/SCID mice. We observed significant extended survival of mice treated with neomycin or neamine. Markers of lymphoma establishment, such as increases in animal body weight, spleen size, tumor cell spleen infiltration, and ascites volume, were observed in nontreated animals and were significantly diminished by neomycin or neamine treatments. A significant decrease in LANA-1 expression, an increase in lytic gene expression, and an increase in cleaved caspase-3 were also observed in neomycin- or neamine-treated animal ascitic cells. These studies demonstrated that ANG played an essential role in KSHV latency maintenance and BCBL-1 cell survival in vivo, and targeting ANG function by neomycin/neamine to induce the apoptosis of cells latently infected with KSHV is an attractive therapeutic strategy against KSHV-associated malignancies.

Uptake of tenofovir and emtricitabine into non-monocytic female genital tract cells with and without hormonal contraceptives.

BackgroundPre-exposure prophylaxis is becoming a strategic component used to control the human immunodeficiency virus (HIV-1) epidemic. The goal of this study was to characterize intracellular uptake of tenofovir and emtricitabine using five surrogate cell lines of the female genital tract and determine whether exogenous hormones influence their uptake.MethodsSurrogate cell lines, ie, THP-1 (representing macrophages), BC-3 (CD8+), Ect1/E6E7 (squamous epithelial), HeLa (CD4+), and TF-1 (dendritic), were incubated for one hour with tenofovir and emtricitabine to assess uptake. In separate experiments, ethinyl estradiol (EE) and etonogestrel (ET) individually and together (EE/ET) were added prior to, simultaneously, and after incubation. Intracellular phosphorylated tenofovir and emtricitabine were quantified using validated tandem mass spectrometry methods.ResultsHeLa and Ect1/E6E7 cells showed significantly increased uptake relative to THP-1 controls for both antiretrovirals. Individually, ethinyl estradiol and etonogestrel significantly altered antiretroviral uptake across all cell lines, except Ect1/E6E7 for tenofovir and HeLa for emtricitabine. Cellular uptake of tenofovir and emtricitabine in BC-3 and TF-1 cells were significantly lower when dosed one hour prior to EE/ET administration compared with each antiretroviral administered in the absence of EE/ET (tenofovir, 80 versus 470 fmol/106 for BC-3 and 77 versus 506 fmol/106 cells for TF-1; emtricitabine, 36 versus 12 fmol/106 for BC-3 and 75 versus 5 fmol/106 cells for TF-1; P < 0.01 for each).ConclusionThese data suggest that intracellular uptake of tenofovir and emtricitabine within the female genital tract varies by cell type and in the presence of hormonal contraceptives. The potential clinical implications of these findings should be further evaluated in vivo.

Role of Human Herpesvirus 8 Interleukin-6-Activated gp130 Signal Transducer in Primary Effusion Lymphoma Cell Growth and Viability

Human herpesvirus 8 (HHV-8) infection is associated with Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease. HHV-8-encoded viral interleukin-6 (vIL-6) is believed to contribute to pathogenesis via proproliferative, antiapoptotic, and proangiogenic activities. In PEL cells, vIL-6 is produced in functional amounts during viral latency and promotes the growth of these cells, mediating its activity from the endoplasmic reticulum (ER), where it is predominantly localized. This vIL-6 activity is dependent, in part, on its interaction with a splice variant of vitamin K epoxide reductase complex subunit 1 (VKORC1), termed VKORC1 variant 2 (VKORC1v2). Here we report that the IL-6 signal transducer, gp130, which can support vIL-6 signaling from the ER, is also required for optimal PEL cell growth and viability. Levels of activated extracellular regulated kinases (ERKs) 1 and 2 and signal transducer and activator of transcription 1 (STAT1) and STAT3, phosphorylated following gp130 stimulation, were reduced in gp130-depleted BCBL-1 and BC-1 cells. Diminished STAT activation was also detected in JSC-1 and BC-3 cells. Effects of gp130 depletion on growth could be mimicked by short hairpin RNA targeting of ERKs 1 and 2 or by depletion of STAT3. Finally, inhibition of vIL-6-gp130 association specifically within the ER compartment suppressed cell proliferation and viability, mirroring the effects of gp130 depletion. Combined, these data demonstrate that gp130, in addition to VKORC1v2, is essential for normal PEL cell growth and survival and that ER-localized vIL-6-gp130 interactions are critical for these activities. Targeting of intracellular vIL-6-gp130 interactions could potentially provide a means of PEL therapy.

HSP70 inhibition by 2-phenylethynesulfonamide induces lysosomal cathepsin D release and immunogenic cell death in primary effusion lymphoma

Heat-shock protein (HSP) 70 is aberrantly expressed in different malignancies and has a cancer-specific cell-protective effect. As such, it has emerged as a promising target for anticancer therapy. In this study, the effect of the HSP70-specific inhibitor (PES), also Pifitrin-μ, on primary effusion lymphoma (PEL) cell viability was analyzed. PES treatment induced a dose- and time-dependent cytotoxic effect in BC3 and BCBL1 PEL cells by inducing lysosome membrane permeabilization, relocation of cathepsin D in the cytosol, Bid cleavage, mitochondrial depolarization with release and nuclear translocation of apoptosis-activating factor. The PES-induced cell death in PEL cells was characterized by the appearance of Annexin-V/propidium iodide double-positive cells from the early times of treatment, indicating the occurrence of an additional type of cell death other than apoptosis, which, accordingly, was not efficiently prevented by the pan-caspase inhibitor Z-VAD-fmk. Conversely, PES-induced cell death was robustly reduced by pepstatin A, which inhibits Bid and caspase 8 processing. In addition, PES was responsible for a block of the autophagic process in PEL cells. Finally, we found that PES-induced cell death has immunogenic potential being able to induce dendritic cell activation.

Abstract 5477: Engineered toxin body demonstrating CD20-specific binding and cell kill in B-cell non-Hodgkin's lymphoma cells.

Abstract Non-Hodgkin's lymphoma (NHL) offers an attractive area for therapeutic biologics. The most successful example of this targeted approach is rituximab, which is approved for front and second line therapy of NHL. Rituximab targets the CD20 antigen expressed on 90% of B-cell NHL cells. However, rituximab has limited direct cell-kill effects, acting instead primarily through indirect immune responses induced by antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC). Direct-cell kill approaches to CD20 have been limited to radioimmunotherapy because CD20 does not efficiently internalize. Despite improved clinical efficacy seen over Rituximab with both 131 tositumomab and ibritumomab tiuxetan, challenges around the administration of radioimmunotherapies have severely curtailed therapeutic use. We have developed an engineered toxin body (ETB) comprising a CD20 scFv and a modified Shiga-like Toxin A (3F7) capable of specifically recognizing and killing CD20 expressing cells. Utilizing a flow cytometry based binding affinity assay, 3F7 demonstrated selective binding to CD20 positive Raji cells whereas no significant binding was observed in CD20 negative BC-1 cells. Further, direct competition of 3F7 binding to Raji cells was observed in the presence of rituximab or the parent monoclonal antibody. 3F7 demonstrated CD20-specific direct cell kill of Raji, Daudi and Ramos cells with minimal cell kill of CD20 negative BC-1 or Jurkat cells. Initial dose finding studies in mice found that a therapeutic dosing schedule was well tolerated. Raji xenograft studies in mice are ongoing. 3F7 is a novel therapeutic approach with direct cell-kill activity against CD20-expressing cells which could expand treatment options for B-cell lymphoma. Citation Format: Rachel Wirth, Sangeetha Rajagopalan, Erin Willert, William Null, Jennifer Erdman, Jen-Sing Liu, Jack P. Higgins. Engineered toxin body demonstrating CD20-specific binding and cell kill in B-cell non-Hodgkin's lymphoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5477. doi:10.1158/1538-7445.AM2013-5477

Preparation and application of polyclonal antibodies against KSHV v-cyclin

We prepared rabbit polyclonal antibodies against Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded v-cyclin (ORF 72) and detected the natural viral protein using these polyclonal antibodies. Three antigenic polypeptides of v-cyclin were designed and synthesized. A fragment of the v-cyclin gene was cloned into a eukaryotic expression vector pEF-MCS-Flag-IRES/Puro to construct a recombinant vector, pEF v-cyclin. Then, pEF v-cyclin was transfected into 293T and EA.hy926 cells to obtain v-cyclin-Flag fusion proteins. Six New Zealand white rabbits were immunized with KLH-conjugated peptides to generate polyclonal antibodies against v-cyclin. The polyclonal antibodies were then characterized by ELISA and Western blotting assays. Finally, the polyclonal antibodies against v-cyclin were used to detect natural viral protein expressed in BCBL-1, BC-3, and JSC-1 cells. The results showed that using the Flag antibody, v-cyclin-Flag fusion protein was detected in 293T and EA.hy926 cells transfected with pEF-v-cyclin. Furthermore, ELISA showed that the titer of the induced polyclonal rabbit anti-v-cyclin antibodies was higher than 1:8,000. In Western blotting assays, the antibodies reacted specifically with the v-cyclin-Flag fusion protein as well as the natural viral protein. The recombinant expression vector pEF-v-cyclin was constructed successfully, and the polyclonal antibodies prepared can be used for various biological tests including ELISA and Western blotting assays.

Reductive Degradation of Organic Compounds Using Microbial Nanotechnology

Members of the genus Clostridia can reduce Pd(II) ions to form metallic Pd nanoparticles (bio-Pd). Cultures of C. pasteurianum BC1 were used to generate bio-Pd, which is primarily formed on the microbial cell wall. Batch experiments using C. pasteurianum BC1 cells loaded with bio-Pd showed efficient reduction of the organic azo dyes, methyl orange and Evans blue, while little reduction of dyes was observed in control experiments using Pd-containing heat-killed microbial cultures or Pd-free viable bacterial cultures. Degradation of azo dyes was found to occur via reductive hydrogenation of the azo-linkage. Molecular hydrogen, which is concomitantly generated by C. pasteurianum, is used in the reduction reaction. The process described in this study is a potentially viable alternative to current groundwater and wastewater treatment technologies that fail to adequately degrade the large quantities of hazardous spent textile dyes that are discharged into the environment each year.