Primary Effusion Lymphoma Cells Research Articles

Evaluating STING Agonist Impact on Kaposi’s Sarcoma-Associated Virus-related Cancers

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an etiological agent of multiple malignancies associated with compromised immunity, such as Kaposi’s sarcoma (KS) and Primary Effusion Lymphoma (PEL). KSHV infection is lifelong with no available treatment or vaccines and poses a high risk to immunocompromised individuals. The virus is transmitted via the oral and sexual route, and has a biphasic life cycle with a short lytic replication cycle followed by long-term latency, with the expression of few genes making detection difficult. Reactivation from latency results in a cascade of viral gene expression and pathogenesis. Critical to controlling KSHV infection is the cGAS-STING DNA-sensing pathway (STING), known to activate the type I interferon response and induce cancer cell death or growth inhibition. STING exhibits both anti-viral and anti-tumor responses, showing promise as a protein of interest to study for the development of a therapeutic treatment. By examining the in vitro effectiveness of STING agonists in inhibiting KSHV oncogenesis in PEL, the study aimed to provide the foundation for a potential novel therapeutic approach. Six patient-derived PEL cell lines were treated with the STING agonist diaminobenzamidazole (diABZI) in a dose-dependent manner. Four of the PEL cell lines exhibited decreased cell growth, viability, and colony formation, while two PEL cell lines were resistant to treatment, correlating with the presence or absence of STING expression. RNA-sequencing was performed and discovered upregulation of multiple interferon-stimulated genes (ISG) in responsive cell lines. Further investigations will explore STING agonists’ inhibition of PEL oncogenesis in vitro via colony formation and in vivo using a PEL xenograft model with NSG mice. Successful completion of this project will offer insight into potential clinical treatments for targeting KSHV infection and reactivation.

The Mitochondrial Ubiquitin Ligase MARCHF5 Cooperates with MCL1 to Inhibit Apoptosis in KSHV-Transformed Primary Effusion Lymphoma Cell Lines.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes several malignancies in people with HIV including Kaposi's sarcoma and primary effusion lymphoma (PEL). We have previously shown that PEL cell lines require myeloid cell leukemia-1 (MCL1) to inhibit apoptosis. MCL1 is an oncogene that is amplified in cancers and causes resistance to chemotherapy regimens. MCL1 is thus an attractive target for drug development. The emerging clinical relevance and therapeutic potential of MCL1 motivated us to study the roles of this oncogene in PEL in depth. Using a systems biology approach, we uncovered an unexpected genetic interaction between MCL1 and MARCHF5 indicating that they function in the same pathway. MARCHF5 is an E3 ubiquitin ligase most known for regulating mitochondrial homeostasis and antiviral signaling, but not apoptosis. We thus investigated how MCL1 and MARCHF5 cooperate to promote PEL cell survival. CRISPR knockout (KO) of MARCHF5 in PEL cell lines resulted in a significant increase in apoptosis despite the presence of MCL1. The anti-apoptotic function of MARCHF5 was dependent on its E3 ligase and dimerization activities. Loss of MARCHF5 or inhibition of the 26S proteasome furthermore stabilized the MCL1 antagonist NOXA without affecting levels of MCL1. Interestingly, NOXA KO provides a fitness advantage to PEL cells suggesting that NOXA is the pro-apoptotic signal that necessitates the anti-apoptotic activities of MCL1 and MARCHF5. Finally, endogenous reciprocal co-immunoprecipitation experiments show that MARCHF5 and NOXA are found in the same protein complex. Our findings thus provide the mechanistic link that underlies the genetic interaction between MCL1 and MARCHF5. We propose that MARCHF5 induces the degradation of the MCL1 antagonist NOXA thus reinforcing the pro-survival role of MCL1 in these tumor cells. This newly appreciated interaction of the MCL1 and MARCHF5 oncogenes may be useful to improve the design of combination therapies for KSHV malignancies.

EZH2 Inhibition by DS3201 Triggers the Kaposi's Sarcoma-Associated Herpesvirus Lytic Cycle and Potentiates the Effects Induced by SAHA in Primary Effusion Lymphoma Cells.

Primary Effusion Lymphoma (PEL) cells carry Kaposi's sarcoma-associated herpesvirus (KSHV) in a latent state, except for a small number of cells in which the virus replicates to ensure its persistence into the infected host. However, the lytic cycle can be reactivated in vitro by exposing these lymphoma cells to various treatments, leading to cell lysis. To restrict viral antigen expression, KSHV induces repressive epigenetic changes, including DNA methylation and histone modifications. Among the latter, histone deacetylation and tri-methylation of Histone H3 lisyne-27 (H3K27me3) have been reported to play a role. Here, we found that the inhibition of H3K27 tri-methylation by valemetostat DS3201 (DS), a small molecule that inhibits Enhancer of Zeste Homolog 2 (EZH2) methyltransferase, induced the KSHV lytic cycle in PEL cells, and that this effect involved the activation of the wtp53-p21 axis and autophagic dysregulation. DS also potentiated the lytic cycle activation mediated by the Histone deacetylases (HDAC) inhibitor Suberoylanilide hydroxamic acid (SAHA) and reinforced its cytotoxic effect, suggesting that such a combination could be used to unbalance the latent/lytic cycle and further impair the survival of PEL cells.

Conserved linear motif within the immediate early protein ORF45 promotes its engagement with KSHV lytic cycle-promoting forkhead transcription factors, FOXK1 and FOXK2.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that can cause several cancers, such as Kaposi sarcoma and primary effusion lymphoma (PEL). We and others have recently demonstrated that Forkhead box (FOX) transcription factors can be dysregulated by KSHV, and they can affect KSHV infection. Herein, we focus on dissecting the role of two FOXK subfamily members, FOXK1 and FOXK2, in the KSHV life cycle. FOXK proteins are key host regulators of cellular functions, yet their role in KSHV infection remains unknown. Here, we demonstrated that both FOXK proteins are essential for efficient KSHV lytic reactivation in PEL cells. FOXK1 and FOXK2 are unique as they are the only FOX proteins that contain a Forkhead-associated (FHA) domain. The FHA domain is a specialized protein binding domain that recognizes a short linear serine/threonine-rich (S/T) motif. Through an unbiased motif survey, we found that KSHV viral protein ORF45 and its gammaherpesvirus homologs contain a putative FHA-binding motif. ORF45 is an immediate early tegument protein, vital for lytic reactivation and virus production. We demonstrated that ORF45 uses its novel conserved motif to interact with the FHA domain containing FOXK factors in the nucleus of infected cells. A single-point mutation of the conserved threonine residue in the motif within ORF45 abolished the ORF45-FOXK1/2 interaction. Our data indicates that FOXK proteins interact with ORF45 homologs encoded by murine gammaherpesvirus 68 (MHV68) and Rhesus macaque rhadinovirus (RRV), and that the FHA domains of FOXK proteins are sufficient for their interactions, highlighting a conserved mechanism.IMPORTANCEThe dysregulation of Forkhead transcription factors contributes to many different human diseases, including cancers, but their impact on herpesvirus lifecycle and pathogenesis is less understood. Our study uncovers a critical pro-lytic function of the FOXK subfamily and its requirement for KSHV lytic reactivation in PEL. We found that FOXK proteins bind to a key immediate early KSHV protein ORF45 using its novel short linear S/T motif. Notably, even though ORF45 homologs in gammaherpesviruses are highly diverse, we identified a similar S/T short linear motif in ORF45 homologs and also showed an evolutionary conserved interaction between FOXK proteins and ORF45 homologs of MHV68 and RRV. Our study provides a basis for future studies in animal models to evaluate the role of FOXK proteins and the impact of their interactions with ORF45 in gammaherpesvirus infection and pathogenesis. Targeting these interactions could allow a novel way to limit gammaherpesvirus infections.

LKB1 suppresses KSHV reactivation and promotes primary effusion lymphoma progression.

Viruses normally reprogram the host cell metabolic pathways as well as metabolic sensors to facilitate their persistence. The serine-threonine liver kinase B1 (LKB1) is a master upstream kinase of 5'-AMP-activated protein kinase (AMPK) that senses the energy status and therefore regulates the intracellular metabolic homeostasis. Previous studies showed that AMPK restricts Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication in endothelial cells during primary infection and promotes primary effusion lymphoma (PEL) cell survival. However, the role of LKB1 in KSHV lytic reactivation and KSHV-associated malignancies is unclear. In this study, we found that LKB1 is phosphorylated or activated in KSHV-positive PEL cells. Mechanistically, KSHV-encoded vCyclin mediated LKB1 activation in PEL cells, as vCyclin knockout ablated, while vCyclin overexpression enhanced LKB1 activation. Furthermore, knockdown of LKB1 inactivated AMPK and induced KSHV reactivation, as indicated by the increased expression of viral lytic genes and the increased virions in supernatants. Accordingly, AMPK inhibition by functional knockdown or a pharmacologic inhibitor, Compound C, promoted KSHV reactivation in PEL cells. Furthermore, inhibition of either LKB1 or AMPKα1 efficiently induced cell death by apoptosis of PEL cells both in vitro and in vivo. Together, these results identify LKB1 as a vulnerable target for PEL, which could be potentially exploited for treating other virus-associated diseases.IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus associated with several human cancers, such as primary effusion lymphoma (PEL). Here, we showed that serine-threonine liver kinase B1 (LKB1), upstream of 5' AMP-activated protein kinase (AMPK), is activated by KSHV-encoded vCyclin and maintains KSHV latency in PEL cells. Inhibition of either LKB1 or AMPK enhances KSHV lytic replication from latency, which at least partially accounts for PEL cell death by apoptosis. Compound C, a potent AMPK inhibitor, induced KSHV reactivation and efficiently inhibited PEL progression in vivo. Thus, our work revealed that LKB1 is a potential therapeutic target for KSHV-associated cancers.

5-AZA Upregulates SOCS3 and PTPN6/SHP1, Inhibiting STAT3 and Potentiating the Effects of AG490 against Primary Effusion Lymphoma Cells.

Epigenetic modifications, including aberrant DNA methylation occurring at the promoters of oncogenes and oncosuppressor genes and histone modifications, can contribute to carcinogenesis. Aberrant methylation mediated by histone methylatransferases, alongside histones, can affect methylation of proteins involved in the regulation of pro-survival pathways such as JAK/STAT and contribute to their activation. In this study, we used DNA or histone demethylating agents, 5-Azacytidine (5-AZA) or DS-3201 (valemetostat), respectively, to treat primary effusion lymphoma (PEL) cells, alone or in combination with AG490, a Signal transducer and activator of transcription 3 (STAT3) inhibitor. Cell viability was investigated by trypan blue assay and FACS analysis. The molecular changes induced by 5-AZA and/or AG490 treatments were investigated by Western blot analysis, while cytokine release by PEL cells treated by these drugs was evaluated by Luminex. Statistical analyses were performed with Graphpad Prism® software (version 9) and analyzed by Student's t test or a nonparametric one-way ANOVA test. The results obtained in this study suggest that 5-AZA upregulated molecules that inhibit STAT3 tyrosine phosphorylation, namely Suppressor of Cytokine Signaling 3 (SOCS3) and tyrosine-protein phosphatase non-receptor type (PTPN) 6/Src homology region 2 domain-containing phosphatase-1 (SHP-1), reducing STAT3 activation and downregulating several STAT3 pro-survival targets in PEL cells. As this lymphoma is highly dependent on the constitutive activation of STAT3, 5-AZA impaired PEL cell survival, and when used in combination with AG490 JAK2/STAT3 inhibitor, it potentiated its cytotoxic effect. Differently from 5-AZA, the inhibition of the EZH1/2 histone methyltransferase by DS-3201, reported to contribute to STAT3 activation in other cancers, slightly affected STAT3 phosphorylation or survival in PEL cells, either alone or in combination with AG490. This study suggests that 5-AZA, by upregulating the expression level of SOCS3 and PTPN6/SHP1, reduced STAT3 activation and improved the outcome of treatment targeting this transcription factor in PEL cells.

Pacritinib inhibits proliferation of primary effusion lymphoma cells and production of viral interleukin-6 induced cytokines

Primary effusion lymphoma (PEL) and a form of multicentric Castleman’s disease (MCD) are both caused by Kaposi sarcoma herpesvirus (KSHV). There is a critical need for improved therapies for these disorders. The IL-6/JAK/STAT3 pathway plays an important role in the pathogenesis of both PEL and KSHV-MCD. We explored the potential of JAK inhibitors for use in PEL and KSHV-MCD, and found that pacritinib was superior to others in inhibiting the growth of PEL cell lines. Pacritinib induced apoptosis in PEL cells and inhibited STAT3 and NF-κB activity as evidenced by reduced amount of phosphorylated moieties. Pacritinib also inhibits FLT3, IRAK1, and ROS1; studies utilizing other inhibitors of these targets revealed that only FLT3 inhibitors exhibited similar cell growth inhibitory effects. FLT3’s likely contribution to pacritinib’s cell growth inhibition was further demonstrated by siRNA knockdown of FLT3. RNA sequencing and RT-PCR showed that many key host genes including cyclins and IL-6 were downregulated by pacritinib, while KSHV genes were variably altered. Finally, pacritinib suppressed KSHV viral IL-6-induced human IL-6 and IL-10 production in peripheral blood mononuclear cells, which may model an important step in KSHV-MCD pathogenesis. These results suggest that pacritinib warrants testing for the treatment of KSHV-MCD and PEL.

Hijacking of nucleotide biosynthesis and deamidation-mediated glycolysis by an oncogenic herpesvirus

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi’s sarcoma (KS) and multiple types of B cell malignancies. Emerging evidence demonstrates that KSHV reprograms host-cell central carbon metabolic pathways, which contributes to viral persistence and tumorigenesis. However, the mechanisms underlying KSHV-mediated metabolic reprogramming remain poorly understood. Carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase (CAD) is a key enzyme of the de novo pyrimidine synthesis, and was recently identified to deamidate the NF-κB subunit RelA to promote aerobic glycolysis and cell proliferation. Here we report that KSHV infection exploits CAD for nucleotide synthesis and glycolysis. Mechanistically, KSHV vCyclin binds to and hijacks cyclin-dependent kinase CDK6 to phosphorylate Ser-1900 on CAD, thereby activating CAD-mediated pyrimidine synthesis and RelA-deamidation-mediated glycolytic reprogramming. Correspondingly, genetic depletion or pharmacological inhibition of CDK6 and CAD potently impeded KSHV lytic replication and thwarted tumorigenesis of primary effusion lymphoma (PEL) cells in vitro and in vivo. Altogether, our work defines a viral metabolic reprogramming mechanism underpinning KSHV oncogenesis, which may spur the development of new strategies to treat KSHV-associated malignancies and other diseases.

Kaposi's sarcoma-associated herpesvirus terminal repeat regulates inducible lytic gene promoters.

The Kaposi's sarcoma-associated herpesvirus (KSHV) genome consists of an approximately 140-kb unique coding region flanked by 30-40 copies of a 0.8-kb terminal repeat (TR) sequence. A gene enhancer recruits transcription-related enzymes by having arrays of transcription factor binding sites. Here, we show that KSHV TR possesses transcription regulatory function with latency-associated nuclear antigen (LANA). Cleavage under targets and release using nuclease demonstrated that TR fragments were occupied by LANA-interacting histone-modifying enzymes in naturally infected cells. The TR was enriched with histone H3K27 acetylation (H3K27Ac) and H3K4 tri-methylation (H3K4me3) modifications and also expressed nascent RNAs. The sites of H3K27Ac and H3K4me3 modifications were also conserved in the KSHV unique region among naturally infected primary effusion lymphoma cells. KSHV origin of lytic replication (Ori-Lyt) showed similar protein and histone modification occupancies with that of TR. In the Ori-Lyt region, the LANA and LANA-interacting proteins colocalized with an H3K27Ac-modified nucleosome along with paused RNA polymerase II. The KSHV transactivator KSHV replication and transcription activator (K-Rta) recruitment sites franked the LANA-bound nucleosome, and reactivation evicted the LANA-bound nucleosome. Including TR fragments in reporter plasmid enhanced inducible viral gene promoter activities independent of the orientations. In the presence of TR in reporter plasmids, K-Rta transactivation was drastically increased, while LANA acquired the promoter repression function. KSHV TR, therefore, functions as an enhancer for KSHV inducible genes. However, in contrast to cellular enhancers bound by multiple transcription factors, perhaps the KSHV enhancer is predominantly regulated by the LANA nuclear body.IMPORTANCEEnhancers are a crucial regulator of differential gene expression programs. Enhancers are the cis-regulatory sequences determining target genes' spatiotemporal and quantitative expression. Here, we show that Kaposi's sarcoma-associated herpesvirus (KSHV) terminal repeats fulfill the enhancer definition for KSHV inducible gene promoters. The KSHV enhancer is occupied by latency-associated nuclear antigen (LANA) and its interacting proteins, such as CHD4. Neighboring terminal repeat (TR) fragments to lytic gene promoters drastically enhanced KSHV replication and transcription activator and LANA transcription regulatory functions. This study, thus, proposes a new latency-lytic switch model in which TR accessibility to the KSHV gene promoters regulates viral inducible gene expression.

HSP110 Inhibition in Primary Effusion Lymphoma Cells: One Molecule, Many Pro-Survival Targets

Heat shock proteins (HSPs) are highly expressed in cancer cells and represent a promising target in anti-cancer therapy. In this study, we investigated for the first time the expression of high-molecular-weight HSP110, belonging to the HSP70 family of proteins, in Primary Effusion Lymphoma (PEL) and explored its role in their survival. This is a rare lymphoma associated with KSHV, for which an effective therapy remains to be discovered. The results obtained from this study suggest that targeting HSP110 could be a very promising strategy against PEL, as its silencing induced lysosomal membrane permeabilization, the cleavage of BID, caspase 8 activation, downregulated c-Myc, and strongly impaired the HR and NHEJ DNA repair pathways, leading to apoptotic cell death. Since chemical inhibitors of this HSP are not commercially available yet, this study encourages a more intense search in this direction in order to discover a new potential treatment that is effective against this and likely other B cell lymphomas that are known to overexpress HSP110.

Corrigendum: Characterization of Human Herpesvirus 8 genomic integration and amplification events in a primary effusion lymphoma cell line

Corrigendum: Characterization of Human Herpesvirus 8 genomic integration and amplification events in a primary effusion lymphoma cell line

Characterization of Human Herpesvirus 8 genomic integration and amplification events in a primary effusion lymphoma cell line

Characterization of Human Herpesvirus 8 genomic integration and amplification events in a primary effusion lymphoma cell line

Targeting FoxO proteins induces lytic reactivation of KSHV for treating herpesviral primary effusion lymphoma.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus consisting of both latent and lytic life cycles. Primary effusion lymphoma (PEL) is an aggressive B-cell lineage lymphoma, dominantly latently infected by KSHV. The latent infection of KSHV is persistent and poses an obstacle to killing tumor cells. Like the "shock and kill" strategy designed to eliminate latent HIV reservoir, methods that induce viral lytic reactivation in tumor latently infected by viruses represent a unique antineoplastic strategy, as it could potentially increase the specificity of cytotoxicity in cancer. Inspired by this conception, we proposed that the induction of KSHV lytic reactivation from latency could be a potential therapeutic stratagem for KSHV-associated cancers. Oxidative stress, the clinical hallmark of PEL, is one of the most prominent inducers for KSHV reactivation. Paradoxically, we found that hydrogen peroxide (H2O2) triggers robust cytotoxic effects on KSHV-negative rather than KSHV-positive B lymphoma cells in a dose-dependent manner. Mechanistically, we identified forkhead box protein O1 (FoxO1) and FoxO3 as irrevocable antioxidant defense genes and both of them are upregulated by KSHV latent infection, which is essential for the promoted ROS scavenging in KSHV-positive B lymphoma cells. Pharmacological inhibition or functional knockdown of either FoxO1 or FoxO3 is sufficient to ablate the antioxidant ability and therefore increases the intracellular ROS level that further reverses KSHV from latency to active lytic replication in PEL cells, resulting in tremendous cell death both in vitro and in vivo. Additionally, the elevated level of ROS by inhibiting FoxO proteins further sensitizes PEL cells to ROS-induced apoptosis. Our study therefore demonstrated that the lytic reactivation of KSHV by inhibiting FoxO proteins is a promising therapeutic approach for PEL, which could be further extended to other virus-associated diseases.

Nigericin Induces Apoptosis in Primary Effusion Lymphoma Cells by Mitochondrial Membrane Hyperpolarization and β-Catenin Destabilization.

Primary effusion lymphoma (PEL) is classified as a rare non-Hodgkin's B-cell lymphoma that is caused by Kaposi's sarcoma-associated herpesvirus (KSHV); PEL cells are latently infected with KSHV. PEL is frequently resistant to conventional chemotherapies. Therefore, the development of novel therapeutic agents is urgently required. Nigericin, a H+ and K+ ionophore, possesses unique pharmacological effects. However, the effects of nigericin on PEL cells remain unknown. We examined the cytotoxic effects of the K+ ionophores, nigericin, nonactin, and valinomycin, on various B-lymphoma cells including PEL. We also evaluated ionophore-induced changes in signaling pathways involved in KSHV-induced oncogenesis. Moreover, the effects of nigericin on mitochondrial membrane potential and viral reactivation in PEL were analyzed. Although the three tested ionophores inhibited the proliferation of several B-lymphoma cell lines, nigericin inhibited the proliferation of PEL cells compared to KSHV-negative cells. In PEL cells, nigericin disrupted the mitochondrial membrane potential and caused the release of cytochrome c, which triggered caspase-9-mediated apoptosis. Nigericin also induced both an increase in phosphorylated p38 MAPK and proteasomal degradation of β-catenin. Combination treatment of nigericin with the p38 MAPK inhibitor SB203580 potentiated the cytotoxic effects towards PEL cells, compared to either compound alone. Meanwhile, nigericin did not influence viral replication in PEL cells. Nigericin induces apoptosis in PEL cells by mitochondrial dysfunction and down-regulation of Wnt/β-catenin signaling. Thus, nigericin is a novel drug candidate for treating PEL without the risk of de novo KSHV infection.

Bcl-xL is required to protect endothelial cells latently infected with KSHV from virus induced intrinsic apoptosis.

Kaposi's Sarcoma herpesvirus (KSHV) is the etiologic agent of Kaposi's Sarcoma (KS), a highly vascularized tumor common in AIDS patients and many countries in Africa. KSHV is predominantly in the latent state in the main KS tumor cell, the spindle cell, a cell expressing endothelial cell markers. To identify host genes important for KSHV latent infection of endothelial cells we previously used a global CRISPR/Cas9 screen to identify genes necessary for the survival or proliferation of latently infected cells. In this study we rescreened top hits and found that the highest scoring gene necessary for infected cell survival is the anti-apoptotic Bcl-2 family member Bcl-xL. Knockout of Bcl-xL or treatment with a Bcl-xL inhibitor leads to high levels of cell death in latently infected endothelial cells but not their mock counterparts. Cell death occurs through apoptosis as shown by increased PARP cleavage and activation of caspase-3/7. Knockout of the pro-apoptotic protein, Bax, eliminates the requirement for Bcl-xL. Interestingly, neither Bcl-2 nor Mcl-1, related and often redundant anti-apoptotic proteins of the Bcl-2 protein family, are necessary for the survival of latently infected endothelial cells, likely due to their lack of expression in all the endothelial cell types we have examined. Bcl-xL is not required for the survival of latently infected primary effusion lymphoma (PEL) cells or other cell types tested. Expression of the KSHV major latent locus alone in the absence of KSHV infection led to sensitivity to the absence of Bcl-xL, indicating that viral gene expression from the latent locus induces intrinsic apoptosis leading to the requirement for Bcl-xL in endothelial cells. The critical requirement of Bcl-xL during KSHV latency makes it an intriguing therapeutic target for KS tumors.

The cellular and KSHV A-to-I RNA editome in primary effusion lymphoma and its role in the viral lifecycle

Adenosine-to-inosine RNA editing is a major contributor to transcriptome diversity in animals with far-reaching biological consequences. Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of several human malignancies including primary effusion lymphoma (PEL). The extent of RNA editing within the KSHV transcriptome is unclear as is its contribution to the viral lifecycle. Here, we leverage a combination of biochemical and genomic approaches to determine the RNA editing landscape in host- and KSHV transcriptomes during both latent and lytic replication in PEL. Analysis of RNA editomes reveals it is dynamic, with increased editing upon reactivation and the potential to deregulate pathways critical for latency and tumorigenesis. In addition, we identify conserved RNA editing events within a viral microRNA and discover their role in miRNA biogenesis as well as viral infection. Together, these results describe the editome of PEL cells as well as a critical role for A-to-I editing in the KSHV lifecycle.

Dihydroartemisinin Induced Apoptosis and Synergized With Chemotherapy in Pleural Effusion Lymphoma Cells.

Primary effusion lymphoma (PEL) is a rare aggressive B-cell lymphoma associated with HHV-8. With a median survival of fewer than six months, the prognosis of the disease with current standard therapies is usually dismal. Dihydroartemisinin (DHA) is a derivative of artemisinin, originally designed as an antimalarial drug. Several studies have shown that this compound also demonstrates anti-cancer activity in various types of cancer, including hematologic malignancies. Anti-proliferation activity of DHA on 5 PEL cell lines was assessed by MTT assay. Cell cycle arrest was determined by propidium iodide staining and flow cytometry analysis. DHA-induced PEL apoptosis was shown by annexin V/PI staining and western blotting for cleaved caspases 3, 8, and 9. An inhibitory effect on PEL growth was evaluated in a PEL-xenograft mouse model. A synergistic effect of DHA and doxorubicin combination treatment was shown in vitro. DHA showed anti-proliferative activity on PEL and induced caspase-dependent apoptosis in a time- and dose-dependent manner. DHA-induced cell death appeared to be triggered by increased levels of reactive oxygen species (ROS). N-acetylcysteine treatment inhibited DHA-induced ROS elevation and suppressed expression of cleaved caspases leading to significantly reduced PEL apoptosis. DHA treatment also demonstrated an inhibitory effect on PEL cell growth in an in-vivo xenograft model. Moreover, we found that a combination treatment of DHA and doxorubicin, the standard chemotherapy drug for PEL, demonstrated a synergistic effect on PEL cell lines. DHA is a potentially effective candidate drug for PEL treatment.

CRISPR screens identify novel regulators of cFLIP dependency and ligand-independent, TRAIL-R1-mediated cell death.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes primary effusion lymphoma (PEL). PEL cell lines require expression of the cellular FLICE inhibitory protein (cFLIP) for survival, although KSHV encodes a viral homolog of this protein (vFLIP). Cellular and viral FLIP proteins have several functions, including, most importantly, the inhibition of pro-apoptotic caspase 8 and modulation of NF-κB signaling. To investigate the essential role of cFLIP and its potential redundancy with vFLIP in PEL cells, we first performed rescue experiments with human or viral FLIP proteins known to affect FLIP target pathways differently. The long and short isoforms of cFLIP and molluscum contagiosum virus MC159L, which are all strong caspase 8 inhibitors, efficiently rescued the loss of endogenous cFLIP activity in PEL cells. KSHV vFLIP was unable to fully rescue the loss of endogenous cFLIP and is therefore functionally distinct. Next, we employed genome-wide CRISPR/Cas9 synthetic rescue screens to identify loss of function perturbations that can compensate for cFLIP knockout. Results from these screens and our validation experiments implicate the canonical cFLIP target caspase 8 and TRAIL receptor 1 (TRAIL-R1 or TNFRSF10A) in promoting constitutive death signaling in PEL cells. However, this process was independent of TRAIL receptor 2 or TRAIL, the latter of which is not detectable in PEL cell cultures. The requirement for cFLIP is also overcome by inactivation of the ER/Golgi resident chondroitin sulfate proteoglycan synthesis and UFMylation pathways, Jagunal homolog 1 (JAGN1) or CXCR4. UFMylation and JAGN1, but not chondroitin sulfate proteoglycan synthesis or CXCR4, contribute to TRAIL-R1 expression. In sum, our work shows that cFLIP is required in PEL cells to inhibit ligand-independent TRAIL-R1 cell death signaling downstream of a complex set of ER/Golgi-associated processes that have not previously been implicated in cFLIP or TRAIL-R1 function.

HSPs/STAT3 Interplay Sustains DDR and Promotes Cytokine Release by Primary Effusion Lymphoma Cells.

Primary effusion lymphoma (PEL) is a rare and aggressive B-cell lymphoma, against which current therapies usually fail. In the present study, we show that targeting HSPs, such as HSP27, HSP70 and HSP90, could be an efficient strategy to reduce PEL cell survival, as it induces strong DNA damage, which correlated with an impairment of DDR. Moreover, as HSP27, HSP70 and HSP90 cross talk with STAT3, their inhibition results in STAT3 de-phosphorylation and. On the other hand, the inhibition of STAT3 may downregulate these HSPs. These findings suggest that targeting HSPs has important implications in cancer therapy, as it can reduce the release of cytokines by PEL cells, which, besides affecting their own survival, could negatively influence anti-cancer immune response.

Application of the CDK9 inhibitor FIT-039 for the treatment of KSHV-associated malignancy

Chronic infection with Kaposi’s sarcoma-associated herpes virus (KSHV) in B lymphocytes causes primary effusion lymphoma (PEL), the most aggressive form of KSHV-related cancer, which is resistant to conventional chemotherapy. In this study, we report that the BCBL-1 KSHV+ PEL cell line does not harbor oncogenic mutations responsible for its aggressive malignancy. Assuming that KSHV viral oncogenes play crucial roles in PEL proliferation, we examined the effect of cyclin-dependent kinase 9 (CDK9) inhibitor FIT-039 on KSHV viral gene expression and KSHV+ PEL proliferation. We found that FIT-039 treatment impaired the proliferation of KSHV+ PEL cells and the expression of KSHV viral genes in vitro. The effects of FIT-039 treatment on PEL cells were further evaluated in the PEL xenograft model that retains a more physiological environment for the growth of PEL growth and KSHV propagation, and we confirmed that FIT-039 administration drastically inhibited PEL growth in vivo. Our current study indicates that FIT-039 is a potential new anticancer drug targeting KSHV for PEL patients.