Primary Effusion Lymphoma Cell Lines Research Articles

Abstract 5110: A novel nucleoside analog therapeutically active against plasma cell malignancies and other ADK-expressing cancers including colon and pancreatic adenocarcinomas

Abstract A number of nucleoside analogues are used successfully for the treatment of several cancers, and in particular leukemias and lymphomas, but these have distinct efficacies for different tumor types, and many malignancies do not respond to currently available nucleoside analogues or other forms of chemotherapy. A high throughput screen conducted in our lab to search for inhibitors of primary effusion lymphoma (PEL) identified the nucleoside analog 6-ethylthioinosine (6-ETI) as a potent and selective inhibitor of PEL, a largely incurable malignancy of B cell origin with plasmacytic differentiation. 6-ETI induced necrosis and ATP-depletion accompanied by S-phase arrest, DNA damage and inhibition of DNA synthesis. To understand 6-ETI mechanism of selectivity, RNA-seq analysis of in vitro generated drug-resistant PEL clones revealed inactivating mutations and loss of expression of adenosine kinase (ADK) as the mechanism of resistance. In vitro assays showed that 6-ETI is a pro-drug that gets phosphorylated and activated by adenosine kinase (ADK) into its active form. We found high ADK expression in PEL cell lines and primary specimens of PEL, multiple myeloma (MM) and plasmablastic lymphoma (PBL) patient samples. 6-ETI was effective at killing multiple myeloma cell lines, primary MM specimens, and had a remarkable anti-tumor response in a disseminated multiple myeloma and PEL xenograft mouse models. Thus, ADK expression can serve as a predictive biomarker to help identify patients that are most likely to respond to 6-ETI treatment. To further assess the spectrum of activity and sensitivity of 6-ETI, we examined ADK expression in other cancer subtypes and found that colorectal and pancreatic adenocarcinomas also overexpress ADK and are highly sensitive to killing by 6-ETI at the low nanomolar concentration. We also found high ADK expression in primary colon and pancreatic adenocarcinoma patient specimens. We compared 6-ETI to other FDA-approved purine analogs and failed to find other compounds with similar potency or selectivity profile. Herein, we report the identification of a novel purine analog, 6-ethylthioinosine, as an effective therapeutic with exquisite sensitivity to plasma cell malignancies and other ADK-expressing cancers. We have successfully used RNASeq-based “resistome” analysis to identify its mechanism of specificity and discovered a new biomarker that can potentially impact patient care and the treatment of some of the most aggressive tumors. Citation Format: Jouliana Sadek, Utthara Nayar, Jonathan Reichel, Jennifer Totonchy, Shizuko Sei, Robert Shoemaker, David Warren, Olivier Elemento, Kenneth Kaye, Ethel Cesarman. A novel nucleoside analog therapeutically active against plasma cell malignancies and other ADK-expressing cancers including colon and pancreatic adenocarcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5110. doi:10.1158/1538-7445.AM2017-5110

Ribonucleotide reductase represents a novel therapeutic target in primary effusion lymphoma.

Primary effusion lymphoma (PEL) is a highly aggressive B-cell malignancy that is closely associated with one of oncogenic viruses infection, Kaposi's sarcoma-associated herpesvirus (KSHV). PEL prognosis is poor and patients barely survive more than 6 months even following active chemotherapy interventions. There is therefore an urgent need to discover more effective targets for PEL management. We recently found that the ribonucleotide reductase (RR) subunit M2 is potentially regulated by the key oncogenic HGF/c-MET pathway in PEL (Dai et al., Blood. 2015;126(26):2821-31). In the current study, we set to investigate the role of RR in PEL pathogenesis and to evaluate its potential as a therapeutic target. We report that the RR inhibitor 3-AP actively induces PEL cell cycle arrest through inhibiting the activity of the NF-κB pathway. Using a xenograft model, we found that 3-AP effectively suppresses PEL progression in immunodeficient mice. Transcriptome analysis of 3-AP treated PEL cell lines reveals altered cellular genes, most of whose roles in PEL have not yet been reported. Taken together, we propose that RR and its signaling pathway may serve as novel actionable targets for PEL management.

YM155 induces apoptosis through proteasome-dependent degradation of MCL-1 in primary effusion lymphoma

Primary effusion lymphoma (PEL) is a lymphoma that shows malignant effusion in body cavities without contiguous tumor masses and has a very poor prognosis. We recently developed a novel drug screening system using patient-derived xenograft (PDX) cells that maintained the primary cell phenotype better than cell lines. This screening is expected to discover anti-tumor drugs that have been overlooked by conventional screening using cell lines. We herein performed this screening to identify new therapeutic agents for PEL.We screened 3518 compounds with known pharmaceutical activities based on cytotoxic effects on PDX cells of PEL and selected YM155, a possible survivin inhibitor. It exerted strong anti-tumor effects in PDX cells and three cell lines of PEL; the GI50 of YM155 was 1.2–7.9nM. We found that YM155 reduced myeloid cell leukemia-1 (MCL-1) protein levels prior to decreasing survivin levels, and this was inhibited by a proteasome inhibitor. The knockdown of MCL-1 by siRNA induced cell death in a PEL cell line, suggesting the involvement of decreased MCL-1 levels in YM155-induced cell death. YM155 also induced the phosphorylation of ERK1/2 and MCL-1, and a MEK1 inhibitor inhibited the phosphorylation of ERK1/2, degradation of MCL-1, and YM155-induced apoptosis. These results indicate that YM155 induces the proteasome-dependent degradation of MCL-1 through its phosphorylation by ERK1/2 and causes apoptosis in PEL cells. Furthermore, a treatment with YM155 significantly inhibited the development of ascites in PEL PDX mice. These results suggest the potential of YM155 as an anti-cancer agent for PEL.

Molecular mechanisms of activating c-MET in KSHV+ primary effusion lymphoma.

The oncogenic Kaposi's sarcoma–associated herpesvirus (KSHV) is a principal causative agent of primary effusion lymphoma (PEL), which is mostly seen in immunosuppressed patients. PEL is a rapidly progressing malignancy with a median survival time of approximately 6 months even under the conventional chemotherapy. We recently report that the hepatocyte growth factor (HGF)/c-MET pathway is highly activated in PEL cells and represents a promising therapeutic target (Blood. 2015;126(26):2821-31). However, the underlying mechanisms of c-MET activation within PEL cells remain largely unknown. To solve this puzzle, here we have utilized the next generation sequencing (NGS) based bioinformatics approach to investigate the genomic landscape of the c-MET gene and we found that there's no single nucleotide variations (SNVs) occurred in the c-MET genomic regions in a cohort of PEL samples. Consistently, Sanger sequencing analysis of frequently mutated exons such as exon 10, 14 and 19 shows no mutation of these c-MET exons in PEL cell-lines, which further supports the notion that mutations are not the major mechanism responsible for c-MET activation in PEL. Further, we found that a transmembrane receptor protein, Plexin-B1, is expressed in PEL cell-lines, which is required for c-MET-mediated PEL cell survival via direct protein interaction.

Role of PU.1 Down-Regulation for the Development of Primary Effusion Lymphoma

Introduction: Primary effusion lymphoma (PEL) is a subtype of aggressive non-Hodgkin lymphoma that shows serous lymphomatous effusion without tumor masses in body cavities especially in advanced acquired immunodeficiency syndrome (AIDS). PEL is universally associated with Kaposi sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 (HHV-8) infection. The prognosis of PEL is extremely poor due to its aggressiveness and resistance to conventional chemotherapy. Therefore, the understanding of the molecular pathogenesis of PEL is vital to develop novel therapeutic strategies. PU.1, one of the Ets family transcription factors, has been reported to be down-regulated in some B-cell malignancies and acute myeloid leukemia. In this study, we examined the methylation of the PU.1 promoter and enhancer regions in PEL cell lines and performed the restoration of PU.1 in PEL cells in vitro and in vivo.Methods:PU.1 mRNA expressions of eight PEL cell lines were examined by polymerase chain reaction (PCR). PU.1 was conditionally expressed using tet-on system in three PEL cell lines. The bisulfite sequencing was performed to analyze the methylation of the PU.1 promoter and enhancer regions in PEL cells. The DNA methyltransferase inhibitor, 5-Azacytidine, was used as an experimental tool for demethylation. The anti-proliferative activities of restoring PU.1 or 5-Azacytidine in PEL cell lines were measured by the methylthiotetrazole method. Apoptosis was quantified using Annexin V antibody. To evaluate the association of PU.1 with TRAIL promoter, we analyzed its recruitment to the promoter by luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay. BCBL-1 cells that expressed tet-inducible PU.1 were injected intraperitoneally into NOD/Rag-2/Jak3double-deficient mice to assess the in vivo efficacy of restoring PU.1. Organ invasion by BCBL-1 cells was evaluated by immunohistochemistry.Results: The expression of PU.1 mRNA in PEL cell lines was weaker than in the human monoblast leukemia cell line, U937, which expresses PU.1 mRNA. The promoter and enhancer regions of the PU.1 gene were highly methylated in PEL cells. The treatment with 5-Azacytidine up-regulated the expression of PU.1 mRNA. Restoration of PU.1 or treatment with 5-Azacytidine inhibited cell proliferation. PU.1 up-regulated TRAIL gene expression among apoptosis-related genes and caused apoptosis. PU.1 induced the transactivation of TRAIL promoter in the reporter assay. Disruption of the PU.1 binding site repressed its transactivation. PU.1 was recruited to TRAIL promoter in the ChIP assay. Although PU.1 induced the master regulator of viral replication, ORF50 expression, the expression of ORF57, which is essential for virus production as a posttranscriptional activator, decreased. PU.1 suppressed ORF 57 by the occupancy of IRF7 in the ORF57 promoter region. Restoration of PU.1 inhibited the ascites formation and organ invasion significantly in vivo compared with control mice.Conclusions: The up-regulation of PU.1 induces growth inhibition and apoptosis via the direct transactivation of TRAIL and modulates viral transactivation via the up-regulation of IRF7. PU.1 is a tumor suppressor in PEL and its down-regulation has a critical role in PEL development. The targeting of PU.1 could be a therapeutic approach in patients with PEL. DisclosuresNo relevant conflicts of interest to declare.

A role for MALT1 activity in Kaposi\u2019s sarcoma-associated herpes virus latency and growth of primary effusion lymphoma

Primary effusion lymphoma (PEL) is an incurable malignancy that develops in immunodeficient patients as a consequence of latent infection of B-cells with Kaposi’s sarcoma-associated herpes virus (KSHV). Malignant growth of KSHV-infected B cells requires the activity of the transcription factor nuclear factor (NF)-κB, which controls maintenance of viral latency and suppression of the viral lytic program. Here we show that the KSHV proteins K13 and K15 promote NF-κB activation via the protease mucosa-associated lymphoid tissue lymphoma translocation protein-1 (MALT1), a key driver of NF-κB activation in lymphocytes. Inhibition of the MALT1 protease activity induced a switch from the latent to the lytic stage of viral infection, and led to reduced growth and survival of PEL cell lines in vitro and in a xenograft model. These results demonstrate a key role for the proteolytic activity of MALT1 in PEL, and provide a rationale for the pharmacological targeting of MALT1 in PEL therapy.

Establishing and characterizing a new primary effusion lymphoma cell line harboring Kaposi's sarcoma-associated herpesvirus.

BackgroundPrimary effusion lymphoma is a rare distinct large B-cell neoplasm that is associated with Kaposi’s sarcoma–associated herpesvirus (KSHV) infection. Over recent years, 9 KSHV-positive/Epstein-Barr virus (EBV)-negative PEL cell lines have been established.MethodsTumor cells were collected from the pleural effusion of a 49-year-old male with AIDS. Cells were grown in RPMI1640 culture medium supplemented with 10 % fetus bovine serum. Single cell cloning was performed successfully by a limiting dilution method in a 96-well plate. The cell line obtained was designated SPEL.ResultsSPEL cells showed gourd-shaped morphology with a polarized nucleus, expressing CD38, CD138, and Blimp-1, but not B cell markers such as CD19 and CD20. Polymerase chain reaction analysis revealed that SPEL cells were positive for KSHV but negative for EBV. Tetradecanoylphorbol acetate induced expression of KSHV lytic proteins and the production of KSHV particles in SPEL cells. Subcutaneous inoculation of SPEL cells into severe combined immunodeficiency mice resulted in the formation of solid tumors. Next-generation sequencing revealed the 138 kbp genome sequence of KSHV in SPEL cells. Suberic bishydroxamate, a histone deacetylase inhibitor, induced the expression of KSHV-encoded lytic proteins and cell death in SPEL cells.ConclusionsA new KSHV-positive and EBV-negative PEL cell line, SPEL was established. This cell line may contribute to furthering our understanding of the pathogenesis of PEL and KSHV infection.

Extracavitary primary effusion lymphoma associated with hemophagocytic lymphohistiocytosis.

Extracavitary primary effusion lymphoma associated with hemophagocytic lymphohistiocytosis.

An Oncogenic Virus Promotes Cell Survival and Cellular Transformation by Suppressing Glycolysis.

Aerobic glycolysis is essential for supporting the fast growth of a variety of cancers. However, its role in the survival of cancer cells under stress conditions is unclear. We have previously reported an efficient model of gammaherpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV)-induced cellular transformation of rat primary mesenchymal stem cells. KSHV-transformed cells efficiently induce tumors in nude mice with pathological features reminiscent of Kaposi’s sarcoma tumors. Here, we report that KSHV promotes cell survival and cellular transformation by suppressing aerobic glycolysis and oxidative phosphorylation under nutrient stress. Specifically, KSHV microRNAs and vFLIP suppress glycolysis by activating the NF-κB pathway to downregulate glucose transporters GLUT1 and GLUT3. While overexpression of the transporters rescues the glycolytic activity, it induces apoptosis and reduces colony formation efficiency in softagar under glucose deprivation. Mechanistically, GLUT1 and GLUT3 inhibit constitutive activation of the AKT and NF-κB pro-survival pathways. Strikingly, GLUT1 and GLUT3 are significantly downregulated in KSHV-infected cells in human KS tumors. Furthermore, we have detected reduced levels of aerobic glycolysis in several KSHV-infected primary effusion lymphoma cell lines compared to a Burkitt’s lymphoma cell line BJAB, and KSHV infection of BJAB cells reduced aerobic glycolysis. These results reveal a novel mechanism by which an oncogenic virus regulates a key metabolic pathway to adapt to stress in tumor microenvironment, and illustrate the importance of fine-tuning the metabolic pathways for sustaining the proliferation and survival of cancer cells, particularly under stress conditions.

Primary B Lymphocytes Infected with Kaposi's Sarcoma-Associated Herpesvirus Can Be Expanded In Vitro and Are Recognized by LANA-Specific CD4+ T Cells.

ABSTRACTKaposi's sarcoma-associated herpesvirus (KSHV) has tropism for B lymphocytes, in which it establishes latency, and can also cause lymphoproliferative disorders of these cells manifesting as primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). T cell immunity is vital for the control of KSHV infection and disease; however, few models of B lymphocyte infection exist to study immune recognition of such cells. Here, we developed a model of B lymphocyte infection with KSHV in which infected tonsillar B lymphocytes were expanded by providing mitogenic stimuli and then challenged with KSHV-specific CD4+ T cells. The infected cells expressed viral proteins found in PELs, namely, LANA and viral IRF3 (vIRF3), albeit at lower levels, with similar patterns of gene expression for the major latency, viral interleukin 6 (vIL-6), and vIRF3 transcripts. Despite low-level expression of open reading frame 50 (ORF50), transcripts for the immune evasion genes K3 and K5 were detected, with some downregulation of cell surface-expressed CD86 and ICAM. The vast majority of infected lymphocytes expressed IgM heavy chains with Igλ light chains, recapitulating the features seen in infected cells in MCD. We assessed the ability of the infected lymphocytes to be targeted by a panel of major histocompatibility complex (MHC) class II-matched CD4+ T cells and found that LANA-specific T cells restricted to different epitopes recognized these infected cells. Given that at least some KSHV latent antigens are thought to be poor targets for CD8+ T cells, we suggest that CD4+ T cells are potentially important effectors for the in vivo control of KSHV-infected B lymphocytes.IMPORTANCE KSHV establishes a latent reservoir within B lymphocytes, but few models exist to study KSHV-infected B cells other than the transformed PEL cell lines, which have likely accrued mutations during the transformation process. We developed a model of KSHV-infected primary B lymphocytes that recapitulates features seen in PEL and MCD by gene expression and cell phenotype analysis, allowing the study of T cell recognition of these cells. Challenge of KSHV-infected B cells with CD4+ T cells specific for LANA, a protein expressed in all KSHV-infected cells and malignancies in vivo, showed that these effectors could efficiently recognize such targets. Given that the virus expresses immune evasion genes or uses proteins with intrinsic properties, such as LANA, that minimize epitope recognition by CD8+ T cells, CD4+ T cell immunity to KSHV may be important for maintaining the virus-host balance.

A potential role of the NOD genetic background in mouse peritoneal macrophages for the development of primary effusion lymphoma

Severe immunodeficient mice have become invaluable tools in human stem cell and tumor research. In this study, we compared the phagocytic activity of peritoneal macrophages against primary effusion lymphoma (PEL) among Rag-2/Jak3 double-deficient (Rag-2−/−Jak3−/−) mice with NOD and non-NOD (Balb/c and C57/BL6). We also evaluated lymphomatous effusion and infiltration in a PEL xenograft mouse model using these severe immunodeficient mice. In the phagocytic assay, peritoneal macrophages in the NOD background phagocytosed CFSE-labeled BCBL-1, a PEL cell line, less efficiently than those in the non-NOD background. BCBL-1 cells were successfully engrafted into both the NOD background and non-NOD background; however, the volume of ascites of the NOD background was significantly higher than that of the non-NOD background. Moreover, the organ invasion of PEL cells was suppressed in non-NOD background mice. Thus, the NOD genetic background is considered to contribute to more lymphomatous effusion and the infiltration of PEL cells than a non-NOD background. Our results showed that the NOD background allowed more lymphomatous effusion and infiltration than other backgrounds and peritoneal macrophages played a critical role in preventing the growth and infiltration of PEL cells.

Exquisite Sensitivity of Plasma Cell Malignancies to a Novel Nucleoside Analog Is Mediated By Overexpressed Adenosine Kinase

Primary effusion lymphoma (PEL) is a largely incurable malignancy of B cell origin, driven by infection with KSHV/HHV-8, and with notable plasmacytic differentiation. PEL carries an extremely poor prognosis with a median survival time of less than 6 months and is known to be largely resistant to conventional chemotherapy. Therefore, we conducted high throughput screens for effective inhibitors using PEL cell lines. We discovered a compound, 6-ethylthioinosine (6-ETI), a nucleoside analog with selective toxicity to PEL and multiple myeloma (MM) at nanomolar concentrations, but not to other lymphoma cell lines tested. 6-ETI also showed remarkable responses in a mouse xenograft model of PEL. To understand the molecular mechanism(s) of selectivity towards PEL, we developed and performed resistome analysis, an unbiased approach based on RNA sequencing of in vitro and in vivo generated resistant subclones. We found adenosine kinase (ADK) inactivating genomic alterations in all resistant clones as the mechanism of resistance. Concordantly, we found that plasma cells express ADK, as well as sensitive PELs and multiple myeloma cell lines, while resistant lymphoma cell lines including those with EBV infection had lower levels (if any); interestingly, the latter could be sensitized by cell crowding-induced ADK upregulation. Like other nucleoside analogues, 6-ETI induces ATP-depletion and cell death accompanied by S-phase arrest and DNA damage, but only in ADK-expressing cells. Immunohistochemistry for ADK served as a new biomarker approach to identify tumors that may be sensitive to 6-ETI, which we documented for primary specimens of PEL, multiple myeloma and other lymphoid malignancies with plasmacytic features, namely plasmablastic lymphoma. A number of nucleoside analogues have been reported to be effective in treating leukemias and B and T cell lymphomas. We performed structure-activity relationship studies and tested a number of nucleoside analogs that are in preclinical or clinical development for other hematological malignancies to identify and better understand the structural requirements for 6-ETIÕs biological activity. We successfully demonstrated that 6-ETI was more potent and selective at killing PEL and MM cell lines than other studied nucleoside analogs suggesting that this compound possesses unique and distinct features that are clinically promising. Despite the presence of treatment approaches that can greatly extended the survival of MM patients, resistance remains an issue, warranting the need for new effective agents and combinations. We identified 6-ETI as a novel and clinically viable nucleoside analog for the treatment of PEL, immunoblastic lymphoma, plasmablastic lymphoma, multiple myeloma and other ADK-expressing cancers. [Display omitted] DisclosuresNayar:Weill Cornell Medical College: Patents & Royalties: Submitted patent applicatio for 6-ETI. Cesarman:Weill Cornell Medical College: Patents & Royalties: applied for patent for 6-ETI.

Selective Inhibitors of Nuclear Export (SINE) Compounds Suppress Both HIV Replication and AIDS Related Lymphoma

Infection with human immunodeficiency virus (HIV) compromises the immune system leaving infected individuals vulnerable to other pathologies including cancer. HIV-infected patients have an increased risk of cancer development as compared to the general population. Primary effusion lymphoma (PEL), a high-grade non-Hodgkin lymphoma with very poor prognosis, typically affects individuals infected with HIV in advanced stages of acquired immune deficiency syndrome (AIDS). While the use of anti-HIV drugs is associated with better prognosis, treating cancer in HIV-infected patients is often challenging due to potential drug interactions between anti-cancer agents and antiretroviral drugs. We demonstrate that inhibition of the exportin-1 (CRM1/XPO1) mediated nuclear protein transport by SINE compounds results in effective suppression of HIV replication, which was quantified by measuring virus-associated core antigen (p24) by ELISA. In addition by using MTT, assays, Annexin staining, FACS and immunoblot analysis we demonstrated that XPO1 inhibition induced cell cycle arrest and apoptosis in PEL cell lines (BC-1, BCBL-1, and JSC-1) at similar doses. SINE blocked the nuclear export of the late viral partially and unspliced RNA species encoding for the viral structural proteins. In BC-1 PEL cells inhibition of XPO1 resulted in the nuclear accumulation of p53 tumor suppressor protein as well as inhibition of NF-κB activity. In vivo, oral administration twice a week for 4 weeks, 20 mg/kg of the clinical SINE compound selinexor (currently in phase I/II clinical trials) led to a significant suppression of the BC-1 PEL growth in vivo. These findings provide a strong basis for inhibiting XPO1 as a novel strategy for the combined treatment of HIV and PEL or other AIDS-related cancers. DisclosuresTamir:Karyopharm: Employment. Kauffman:Karyopharm: Employment, Equity Ownership. Shacham:Karyopharm: Employment, Equity Ownership. Landesman:Karyopharm: Employment.

PEGylated siRNA lipoplexes for silencing of BLIMP-1 in Primary Effusion Lymphoma: In vitro evidences of antitumoral activity

Silencing of the B lymphocyte-induced maturation protein 1 (Blimp-1), a pivotal transcriptional regulator during terminal differentiation of B cells into plasma cells with siRNAs is under investigation as novel therapeutic approach in Primary Effusion Lymphoma (PEL), a HHV-8 related and aggressive B cell Lymphoma currently lacking of an efficacious therapeutic approach.The clinical application of small interfering RNA (siRNA) in cancer therapy is limited by the lack of an efficient systemic siRNA delivery system.In this study we aim to develop pegylated siRNA lipoplexes formed using the cationic lipid DOTAP and DSPE-PEG2000, capable to effectively stabilize anti-Blimp-1 siRNA and suitable for systemic administration.Two types of pegylated lipoplexes using a classic (C-PEG Lipoplexes) or a post-pegylation method (P-PEG-Lipoplexes) were formulated and compared in their physicochemical properties (size, zeta potential, morphology and structure) and efficiency on PEL cell lines.A stable siRNAs protection was obtained with post pegylation approach (2% molar of DSPE-PEG2000 with respect to lipid) resulting in structures with diameters of 300nm and a complexation efficiency higher that 80% (0.08nmol/10nmol of lipid). In vitro studies on PEL cell lines suggested that empty liposomes were characterized by a low cell toxicity also after PEG modification (cell viability and cell density over 85% after treatment with 10μM of lipid).We demonstrated that P-PEG-Lipoplexes were able to significantly reduce the levels of BLIMP-1 protein leading to reduction of viability (less that 15% after transfection with 100nM of complexed siRNAs) and activation of apoptosis.In vitro efficiency encourages us to further test the in vivo potential of P-PEG-Lipoplexes in PEL therapy.

Constitutive Activation of Interleukin-13/STAT6 Contributes to Kaposi's Sarcoma-Associated Herpesvirus-Related Primary Effusion Lymphoma Cell Proliferation and Survival.

Activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway has been associated with numerous human malignancies, including primary effusion lymphomas (PELs). PEL, a cancerous proliferation of B cells, is caused by Kaposi's sarcoma-associated herpesvirus (KSHV). Previously we identified constitutive phosphorylation of STAT6 on tyrosine 641 (p-STAT6(C)) in PEL cell lines BC3 and BCBL1; however, the molecular mechanism leading to this activation remains unclear. Here we demonstrate that STAT6 activation tightly correlates with interleukin-13 (IL-13) secretion, JAK1/2 tyrosine phosphorylation, and reduced expression of SHP1 due to KSHV infection. Moreover, p-STAT6(C) and reduction of SHP1 were also observed in KS patient tissue. Notably, blockade of IL-13 by antibody neutralization dramatically inhibits PEL cell proliferation and survival. Taken together, these results suggest that IL-13/STAT6 signaling is modulated by KSHV to promote host cell proliferation and viral pathogenesis. STAT6 is a member of signal transducer and activator of transcription (STAT) family, whose activation is linked to KSHV-associated cancers. The mechanism through which STAT6 is modulated by KSHV remains unclear. In this study, we demonstrated that constitutive activation of STAT6 in KSHV-associated PEL cells results from interleukin-13 (IL-13) secretion and reduced expression of SHP1. Importantly, we also found that depletion of IL-13 reduces PEL cell growth and survival. This discovery provides new insight that IL-13/STAT6 plays an essential role in KSHV pathogenesis.

Abstract 4496: Genomics-based resistome analysis revealed endogenous adenosine kinase levels as a chief determinant of specificity for a novel nucleoside analog lymphoma inhibitor

Abstract We used KSHV-infected primary effusion lymphoma (PEL) cells as a model to search for novel drugs through a combination of screening and genomic approaches. We discovered 6-ethylthioinosine (6-ETI), a nucleoside analog, as a highly effective and selective inhibitor of PEL. This compound induces necrosis accompanied by S-phase arrest without affecting known oncogenic viral proteins. To understand the selectivity towards PEL, we performed unbiased genomic analysis of 6-ETI-resistant subclones using RNASeq, which revealed that 6-ETI is activated through phosphorylation by cellular adenosine kinase (ADK). PEL cell lines have higher basal levels of ADK than resistant lymphoma cell lines, and resistant lymphoma cell lines could be sensitized by cell crowding-induced ADK upregulation. Finally, 6-ETI was shown to be highly efficacious against incipient and established tumors in a mouse model of PEL, with no apparent toxicity. Thus, we have successfully used RNASeq-based “resistome” analysis to identify the mechanism of specificity for a new and preclinically effective nucleoside analog. Citation Format: Utthara Nayar, Jonathan Reichel, Jouliana Sadek, Denise Hernandez-Hopkins, Gunkut Akar, Hufeng Zhou, Michelle A. Sahai, Peter Barelli, Ilaria Guasparri, Jennifer Totonchy, Duane Hassane, Shizuko Sei, Robert H. Shoemaker, J. David Warren, Olivier Elemento, Kenneth M. Kaye, Ethel Cesarman. Genomics-based resistome analysis revealed endogenous adenosine kinase levels as a chief determinant of specificity for a novel nucleoside analog lymphoma inhibitor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4496. doi:10.1158/1538-7445.AM2015-4496

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 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.

Fumagillin, a potent angiogenesis inhibitor, induces Kaposi sarcoma-associated herpesvirus replication in primary effusion lymphoma cells

Kaposi sarcoma and primary effusion lymphoma cells are infected with Kaposi sarcoma-associated herpesvirus (KSHV), predominantly in the latent form, and KSHV replication is observed rarely. Angiogenesis plays a crucial role in the pathogenesis of both Kaposi sarcoma and primary effusion lymphoma. In this study, we found that fumagillin, a potent angiogenesis inhibitor, induced replication of KSHV in primary effusion lymphoma cell lines. The transcript and protein product of replication transcriptional activator (RTA) were induced by 1–10 μM fumagillin at 24 and 48 h, respectively. Western blot analysis demonstrated that 10 μM fumagillin induced not only RTA expression but also other KSHV-encoded lytic proteins. A real-time PCR array detecting KSHV gene expression demonstrated that the expression profiles of KSHV induced by fumagillin were similar to those induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), but the amounts of each transcript were lower than those induced by TPA. Finally, real-time PCR demonstrated an increase in that viral DNA copy number per cell in fumagillin-stimulated primary effusion lymphoma cell lines, indicating replication of KSHV. In addition to TPA, 10 μM fumagillin resulted in growth inhibition of primary effusion lymphoma cell lines. These observations suggest that an angiogenesis inhibitor is an agent with potent effects on cell growth and KSHV reactivation in primary effusion lymphoma cells.

Activated Nrf2 Interacts with Kaposi's Sarcoma-Associated Herpesvirus Latency Protein LANA-1 and Host Protein KAP1 To Mediate Global Lytic Gene Repression

Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease. We have previously shown that KSHV utilizes the host transcription factor Nrf2 to aid in infection of endothelial cells and oncogenesis. Here, we investigate the role of Nrf2 in PEL and PEL-derived cell lines and show that KSHV latency induces Nrf2 protein levels and transcriptional activity through the COX-2/PGE2/EP4/PKCζ axis. Next-generation sequencing of KSHV transcripts in the PEL-derived BCBL-1 cell line revealed that knockdown of this activated Nrf2 results in global elevation of lytic genes. Nrf2 inhibition by the chemical brusatol also induces lytic gene expression. Both Nrf2 knockdown and brusatol-mediated inhibition induced KSHV lytic reactivation in BCBL-1 cells. In a series of follow-up experiments, we characterized the mechanism of Nrf2-mediated regulation of KSHV lytic repression during latency. Biochemical assays showed that Nrf2 interacted with KSHV latency-associated nuclear antigen 1 (LANA-1) and the host transcriptional repressor KAP1, which together have been shown to repress lytic gene expression. Promoter studies showed that although Nrf2 alone induces the open reading frame 50 (ORF50) promoter, its association with LANA-1 and KAP1 abrogates this effect. Interestingly, LANA-1 is crucial for efficient KAP1/Nrf2 association, while Nrf2 is essential for LANA-1 and KAP1 recruitment to the ORF50 promoter and its repression. Overall, these results suggest that activated Nrf2, LANA-1, and KAP1 assemble on the ORF50 promoter in a temporal fashion. Initially, Nrf2 binds to and activates the ORF50 promoter during early de novo infection, an effect that is exploited during latency by LANA-1-mediated recruitment of the host transcriptional repressor KAP1 on Nrf2. Cell death assays further showed that Nrf2 and KAP1 knockdown induce significant cell death in PEL cell lines. Our studies suggest that Nrf2 modulation through available oral agents is a promising therapeutic approach in the treatment of KSHV-associated malignancies. KS and PEL are aggressive KSHV-associated malignancies with moderately effective, highly toxic chemotherapies. Other than ganciclovir and alpha interferon (IFN-α) prophylaxis, no KSHV-associated chemotherapy targets the underlying infection, a major oncogenic force. Hence, drugs that selectively target KSHV infection are necessary to eradicate the malignancy while sparing healthy cells. We recently showed that KSHV infection of endothelial cells activates the transcription factor Nrf2 to promote an environment conducive to infection and oncogenesis. Nrf2 is modulated through several well-tolerated oral agents and may be an important target in KSHV biology. Here, we investigate the role of Nrf2 in PEL and demonstrate that Nrf2 plays an important role in KSHV gene expression, lytic reactivation, and cell survival by interacting with the host transcriptional repressor KAP1 and the viral latency-associated protein LANA-1 to mediate global lytic gene repression and thus cell survival. Hence, targeting Nrf2 with available therapies is a viable approach in the treatment of KSHV malignancies.

Nutlin-3 loaded nanocarriers: Preparation, characterization and in vitro antineoplastic effect against primary effusion lymphoma

In this investigation, Nutlin-3 (Nut3), a novel antitumor drug with low water solubility (<0.1mg/L at 25°C), was loaded into liposomes (Lipo-Nut3), polymeric nanoparticles (NPs-Nut3) and nanoparticles engineered with an antibody direct against Syndecan-1/CD 138 (Syn-NPs-Nut3) to obtain carriers targeted to PEL (primary effusion lymphoma). The physicochemical properties of these carriers were determined. Atomic force microscopy showed that all the particles were well formed and spherical in shape. The presence of the antibody on surface led to a significant increase of mean diameter (280±63nm), PDI (0.3) and the shift of zeta potential towards neutrality (−1mV). The entrapment efficiency of Lipo-Nut3, NPs-Nut3 and Syn-NPs-Nut3 was 30, 52 and 29%, and drug loading was 1.4, 4.5 and 2.6%, respectively. By performing cytofluorimetric analyses and bromodeoxyuridine (BrdU) assay, the efficacy of nanocarriers to deliver the antineoplastic drug into a PEL cell line namely BCBL-1 (immortalized body cavity B-cell lymphoma) was investigated. Two days after the treatment with 20μM of Syn-NPs-Nut3, the cell density decreased at about 60% while the cell viability decreased at 56% only 5 days after transfection, when compared with untreated cells. A cell cycle arrest was observed with a significant decrease of cells in S-phase and increasing of apoptotic cell, if compared with untreated control. These results confirms the potential of nanocarriers approaches to deliver antitumor drug with unfavorable chemico-physical properties. Moreover, this study strongly suggests that Syn-NPs-Nut3 can be a valuable drug carrier system for the treatment of PEL lymphoma.