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Kaposi's sarcoma herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS), a hyperplasia consisting of enlarged malformed vasculature and spindle-shaped cells, the main proliferative component of KS. While spindle cells express markers of lymphatic and blood endothelium, the origin of spindle cells is unknown. Endothelial precursor cells have been proposed as the source of spindle cells. We previously identified two types of circulating endothelial colony forming cells (ECFCs), ones that expressed markers of blood endothelium and ones that expressed markers of lymphatic endothelium. Here we examined both blood and lymphatic ECFCs infected with KSHV. Lymphatic ECFCs are significantly more susceptible to KSHV infection than the blood ECFCs and maintain the viral episomes during passage in culture while the blood ECFCs lose the viral episome. Only the KSHV-infected lymphatic ECFCs (K-ECFCLY) grew to small multicellular colonies in soft agar whereas the infected blood ECFCs and all uninfected ECFCs failed to proliferate. The K-ECFCLYs express high levels of SOX18, which supported the maintenance of high copy number of KSHV genomes. When implanted subcutaneously into NSG mice, the K-ECFCLYs persisted in vivo and recapitulated the phenotype of KS tumor cells with high number of viral genome copies and spindling morphology. These spindle cell hallmarks were significantly reduced when mice were treated with SOX18 inhibitor, SM4. These data suggest that KSHV-infected lymphatic ECFCs can be utilized as a KSHV infection model for in vivo translational studies to test novel inhibitors representing potential treatment modalities for KS., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Gertrude Biomedical Pty Ltd. participated in the study design, analysis, decision to publish, and preparation of the manuscript., (Copyright: © 2023 Tuohinto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

CRISPR activation (CRISPRa) has revealed great potential as a tool to modulate the expression of targeted cellular genes. Here, we successfully applied the CRISPRa system to trigger the Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation in latently infected cells by selectively activating ORF50 gene directly from the virus genome. We found that a nuclease-deficient Cas9 (dCas9) fused to a destabilization domain (DD) and 12 copies of the VP16 activation domain (VP192) triggered a more efficient KSHV lytic cycle and virus production when guided to two different sites on the ORF50 promoter, instead of only a single site. To our surprise, the virus reactivation induced by binding of the stable DD-dCas9-VP192 on the ORF50 promoter was even more efficient than reactivation induced by ectopic expression of ORF50. This suggests that recruitment of additional transcriptional activators to the ORF50 promoter, in addition to ORF50 itself, are needed for the efficient virus production. Further, we show that CRISPRa can be applied to selectively express the early lytic gene, ORF57, without disturbing the viral latency. Therefore, CRISPRa-based systems can be utilized to facilitate virus-host interaction studies by controlling the expression of not only cellular but also of specific KSHV genes.

Kaposi sarcoma is a tumor caused by Kaposi sarcoma herpesvirus (KSHV) infection and is thought to originate from lymphatic endothelial cells (LEC). While KSHV establishes latency in virtually all susceptible cell types, LECs support spontaneous expression of oncogenic lytic genes, high viral genome copies, and release of infectious virus. It remains unknown the contribution of spontaneous virus production to the expansion of KSHV-infected tumor cells and the cellular factors that render the lymphatic environment unique to KSHV life cycle. We show here that expansion of the infected cell population, observed in LECs, but not in blood endothelial cells, is dependent on the spontaneous virus production from infected LECs. The drivers of lymphatic endothelium development, SOX18 and PROX1, regulated different steps of the KSHV life cycle. SOX18 enhanced the number of intracellular viral genome copies and bound to the viral origins of replication. Genetic depletion or chemical inhibition of SOX18 caused a decrease of KSHV genome copy numbers. PROX1 interacted with ORF50, the viral initiator of lytic replication, and bound to the KSHV genome in the promoter region of ORF50, increasing its transactivation activity and KSHV spontaneous lytic gene expression and infectious virus release. In Kaposi sarcoma tumors, SOX18 and PROX1 expression correlated with latent and lytic KSHV protein expression. These results demonstrate the importance of two key transcriptional drivers of LEC fate in the regulation of the tumorigenic KSHV life cycle. Moreover, they introduce molecular targeting of SOX18 as a potential novel therapeutic avenue in Kaposi sarcoma. SIGNIFICANCE: SOX18 and PROX1, central regulators of lymphatic development, are key factors for KSHV genome maintenance and lytic cycle in lymphatic endothelial cells, supporting Kaposi sarcoma tumorigenesis and representing attractive therapeutic targets., (©2020 American Association for Cancer Research.)

Kaposi's sarcoma (KS) is an endothelial tumor causally linked to Kaposi's sarcoma herpesvirus (KSHV) infection. At early stages of KS, inflammation and aberrant neoangiogenesis are predominant, while at late stages the disease is characterized by the proliferation of KSHV-infected spindle cells (SC). Since KSHV infection modifies the endothelial cell (EC) identity, the origin of SCs remains elusive. Yet, pieces of evidence indicate the lymphatic origin. KSHV-infected ECs display increased proliferative, angiogenic and migratory capacities which account for KS oncogenesis. Here we propose a model in which KSHV reprograms the EC identity, induces DNA damage and establishes a dysregulated gene expression program involving interplay of latent and lytic genes allowing continuous reinfection of ECs attracted to the tumor by the secretion of virus-induced cellular factors., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Classic Kaposi sarcoma (cKS) is an inflammatory tumor caused by human herpesvirus 8 (HHV-8) commonly observed in elderly men of Mediterranean origin. We studied a Finnish family of 5 affected individuals in 2 generations. Except for atypical mycobacterial infection of the index case, the affected individuals did not have notable histories of infection., Methods: We performed genome and exome sequencing and mapped shared chromosomal regions to identify genetic predisposition in the family., Results: We identified 12 protein-coding candidate variants that segregated in the 3 affected cousins from whom we had samples. The affected mother of the index case was an obligatory carrier. Among the 12 candidates was a rare heterozygous substitution rs141331848 (c.1337C>T, p.Thr446Ile) in the DNA-binding domain of STAT4. The variant was not present in 242 Finnish control genomes or 180 additional regional controls. Activated T-helper cells from the HHV-8-negative variant carriers showed reduced interferon γ production, compared with age and sex matched wild-type individuals. We screened STAT4 in additional 18 familial KS cases and the variant site from 56 sporadic KS cases but detected no pathogenic mutations., Conclusions: Our data suggest that STAT4 is a potential cKS-predisposition gene, but further functional and genetic validation is needed., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Kaposi sarcoma (KS), an angioproliferative disease associated with Kaposi sarcoma herpesvirus (KSHV) infection, harbors a diversity of cell types ranging from endothelial to mesenchymal cells of unclear origin. We developed a three-dimensional cell model for KSHV infection and used it to demonstrate that KSHV induces transcriptional reprogramming of lymphatic endothelial cells to mesenchymal cells via endothelial-to-mesenchymal transition (EndMT). KSHV-induced EndMT was initiated by the viral proteins vFLIP and vGPCR through Notch pathway activation, leading to gain of membrane-type-1 matrix metalloproteinase (MT1-MMP)-dependent invasive properties and concomitant changes in viral gene expression. Mesenchymal markers and MT1-MMP were found codistributed with a KSHV marker in the same cells from primary KS biopsies. Our data explain the heterogeneity of cell types within KS lesions and suggest that KSHV-induced EndMT may contribute to KS development by giving rise to infected, invasive cells while providing the virus a permissive cellular microenvironment for efficient spread., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Nucleophosmin (NPM) is a multifunctional nuclear phosphoprotein and a histone chaperone implicated in chromatin organization and transcription control. Oncogenic Kaposi's sarcoma herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). In the infected host cell KSHV displays two modes of infection, the latency and productive viral replication phases, involving extensive viral DNA replication and gene expression. A sustained balance between latency and reactivation to the productive infection state is essential for viral persistence and KSHV pathogenesis. Our study demonstrates that the KSHV v-cyclin and cellular CDK6 kinase phosphorylate NPM on threonine 199 (Thr199) in de novo and naturally KSHV-infected cells and that NPM is phosphorylated to the same site in primary KS tumors. Furthermore, v-cyclin-mediated phosphorylation of NPM engages the interaction between NPM and the latency-associated nuclear antigen LANA, a KSHV-encoded repressor of viral lytic replication. Strikingly, depletion of NPM in PEL cells leads to viral reactivation, and production of new infectious virus particles. Moreover, the phosphorylation of NPM negatively correlates with the level of spontaneous viral reactivation in PEL cells. This work demonstrates that NPM is a critical regulator of KSHV latency via functional interactions with v-cyclin and LANA.

KSHV infection is the causative agent in three different tumor types: Kaposi's sarcoma, a plasmablastic variant of multicentric Castelman's disease and an AIDS-related form of B cell lymphoproliferative disorder called primary effusion lymphoma (PEL). PEL manifests as an effusion malignancy in Kaposi's sarcoma patients with advanced AIDS, but also occurs in HIV-negative individuals. PEL is a very aggressive disease, and currently there are no efficient therapies for treating PEL. In our recent paper we report that p53 reactivation by a small molecule inhibitor of p53-MDM2 interaction, Nutlin-3a, induces selective and massive apoptosis in PEL cells, and has striking anti-tumor activity in a mouse xenograft PEL model. In the light of current treatment regimens for PEL, we discuss here the benefits of using reactivation of the p53 pathway as a novel principle for the treatment of this virally induced highly aggressive malignancy.

Kaposi sarcoma is a tumor consisting of Kaposi sarcoma herpesvirus (KSHV)-infected tumor cells that express endothelial cell (EC) markers and viral genes like v-cyclin, vFLIP, and LANA. Despite a strong link between KSHV infection and certain neoplasms, de novo virus infection of human primary cells does not readily lead to cellular transformation. We have studied the consequences of expression of v-cyclin in primary and immortalized human dermal microvascular ECs. We show that v-cyclin, which is a homolog of cellular D-type cyclins, induces replicative stress in ECs, which leads to senescence and activation of the DNA damage response. We find that antiproliferative checkpoints are activated upon KSHV infection of ECs, and in early-stage but not late-stage lesions of clinical Kaposi sarcoma specimens. These are some of the first results suggesting that DNA damage checkpoint response also functions as an anticancer barrier in virally induced cancers.

Kaposi's sarcoma herpesvirus (KSHV) is the etiologic agent for primary effusion lymphoma (PEL), a non-Hodgkin type lymphoma manifesting as an effusion malignancy in the affected individual. Although KSHV has been recognized as a tumor virus for over a decade, the pathways for its tumorigenic conversion are incompletely understood, which has greatly hampered the development of efficient therapies for KSHV-induced malignancies like PEL and Kaposi's sarcoma. There are no current therapies effective against the aggressive, KSHV-induced PEL. Here we demonstrate that activation of the p53 pathway using murine double minute 2 (MDM2) inhibitor Nutlin-3a conveyed specific and highly potent activation of PEL cell killing. Our results demonstrated that the KSHV latency-associated nuclear antigen (LANA) bound to both p53 and MDM2 and that the MDM2 inhibitor Nutlin-3a disrupted the p53-MDM2-LANA complex and selectively induced massive apoptosis in PEL cells. Together with our results indicating that KSHV-infection activated DNA damage signaling, these findings contribute to the specificity of the cytotoxic effects of Nutlin-3a in KSHV-infected cells. Moreover, we showed that Nutlin-3a had striking antitumor activity in vivo in a mouse xenograft model. Our results therefore present new options for exploiting reactivation of p53 as what we believe to be a novel and highly selective treatment modality for this virally induced lymphoma.

Kaposi's sarcoma herpesvirus (KSHV) is the eighth human herpesvirus discovered in 1994 from Kaposi's sarcoma lesion of an AIDS patient. The strong molecular and epidemiological links associating KSHV with Kaposi's sarcoma and certain lymphoproliferative disorders indicate that KSHV is required for the development of these malignancies. Although KSHV is equipped to manipulate and deregulate several cellular signaling pathways, it is not yet understood how this leads to cell transformation. Profound understanding of the interplay of viral and cellular factors in KSHV-infected cells will provide valuable information on the mechanisms of viral tumorigenesis and enable development of efficient targeted therapies for virus-induced cancers. This review focuses on the cellular signaling pathways that KSHV gene products impinge on and discusses their putative contribution to tumorigenesis.

Kaposi sarcoma herpesvirus (KSHV) infection is consistently associated with primary effusion lymphomas (PELs) that are non-Hodgkin lymphomas of B-cell origin. All PEL cells are latently infected with KSHV and express latent viral proteins such as the viral cyclin (v-cyclin), which has previously been implicated in down-regulation of cell-cycle inhibitor p27(KIP1) levels via phosphorylation on Thr187. PEL cells retain high levels of p27(KIP1) but yet proliferate actively, which has left the biologic significance of this p27(KIP1) destabilization somewhat elusive. We have recently demonstrated that v-cyclin and p27(KIP1) stably associate in PEL cells. Here we demonstrate that v-cyclin together with its kinase partner CDK6 phosphorylates the associated p27(KIP1) in PEL cells, which represent a biologically relevant model system for KSHV pathobiology. During latent viral replication p27(KIP1) was phosphorylated by v-cyclin-CDK6 predominantly on Ser10, which enhances its cytoplasmic localization. Interestingly, upon reactivation of KSHV lytic cycle, v-cyclin-CDK6 phosphorylated p27(KIP1) on Thr187, which resulted in down-regulation of p27(KIP1) protein levels. These findings indicate that v-cyclin modulates the cell-cycle inhibitory function of p27(KIP1) by phosphorylation in PELs, and also suggest a novel role for v-cyclin in the lytic reactivation of KSHV.

Primary effusion lymphomas (PELs) represent a unique non-Hodgkin lymphoma that is consistently infected by Kaposi sarcoma herpesvirus (KSHV). PEL cells express high levels of the cell cycle inhibitor p27(KIP1) and yet proliferate actively. KSHV genome encodes a viral cyclin homolog, v-cyclin, which has previously been implicated in down-regulation of p27(KIP1) levels. To address how PEL cells can tolerate high p27(KIP1) levels, we investigated functional interactions between v-cyclin and p27(KIP1) using PEL-derived cell lines as a model system. Here we demonstrate that v-cyclin and p27(KIP1) stably associate in PEL cells in vivo suggesting an attractive model by which p27(KIP1) is inactivated in the actively proliferating PEL cells. Moreover, we show that v-cyclin and cyclin-dependent kinase 6 (CDK6) form an active kinase without p27(KIP1) and that CDK6 is the in vivo catalytic subunit of v-cyclin in PEL cells. These findings suggest that KSHV may promote oncogenesis in PEL by expressing v-cyclin, which both overrides negative cell cycle controls present in the PEL precursor cells and induces a strong proliferative signal via CDK6 kinase activity.