High-throughput sequencing analysis of a "hit and run" cell and animal model of KSHV tumorigenesis.

Kaposi's sarcoma (KS), is an AIDS-associated neoplasm caused by the KS herpesvirus (KSHV/ HHV-8). KSHV-induced sarcomagenesis is the consequence of oncogenic viral gene expression as well as host genetic and epigenetic alterations. Although KSHV is found in all KS-lesions, the percentage of KSHV-infected (LANA+) spindle-cells of the lesion is variable, suggesting the existence of KS-spindle cells that have lost KSHV and proliferate autonomously or via paracrine mechanisms. A mouse model of KSHVBac36-driven tumorigenesis allowed us to induce KSHV-episome loss before and after tumor development. Although infected cells that lose the KSHV-episome prior to tumor formation lose their tumorigenicity, explanted tumor cells that lost the KSHV-episome remained tumorigenic. This pointed to the existence of virally-induced irreversible oncogenic alterations occurring during KSHV tumorigenesis supporting the possibility of hit and run viral-sarcomagenesis. RNA-sequencing and CpG-methylation analysis were performed on KSHV-positive and KSHV-negative tumors that developed following KSHV-episome loss from explanted tumor cells. When KSHV-positive cells form KSHV-driven tumors, along with viral-gene upregulation there is a tendency for hypo-methylation in genes from oncogenic and differentiation pathways. In contrast, KSHV-negative tumors formed after KSHV-episome loss, show a tendency towards gene hyper-methylation when compared to KSHV-positive tumors. Regarding occurrence of host-mutations, we found the same set of innate-immunity related mutations undetected in KSHV-infected cells but present in all KSHV-positive tumors occurring en exactly the same position, indicating that pre-existing host mutations that provide an in vivo growth advantage are clonally-selected and contribute to KSHV-tumorigenesis. In addition, KSHV-negative tumors display de novo mutations related to cell proliferation that, together with the PDGFRAD842V and other proposed mechanism, could be responsible for driving tumorigenesis in the absence of KSHV-episomes. KSHV-induced irreversible genetic and epigenetic oncogenic alterations support the possibility of "hit and run" KSHV-sarcomagenesis and point to the existence of selectable KSHV-induced host mutations that may impact AIDS-KS treatment.

PDGFRA defines the mesenchymal stem cell Kaposi's sarcoma progenitors by enabling KSHV oncogenesis in an angiogenic environment.

Kaposi's sarcoma (KS) is an AIDS-defining cancer caused by the KS-associated herpesvirus (KSHV). Unanswered questions regarding KS are its cellular ontology and the conditions conducive to viral oncogenesis. We identify PDGFRA(+)/SCA-1(+) bone marrow-derived mesenchymal stem cells (Pα(+)S MSCs) as KS spindle-cell progenitors and found that pro-angiogenic environmental conditions typical of KS are critical for KSHV sarcomagenesis. This is because growth in KS-like conditions generates a de-repressed KSHV epigenome allowing oncogenic KSHV gene expression in infected Pα(+)S MSCs. Furthermore, these growth conditions allow KSHV-infected Pα(+)S MSCs to overcome KSHV-driven oncogene-induced senescence and cell cycle arrest via a PDGFRA-signaling mechanism; thus identifying PDGFRA not only as a phenotypic determinant for KS-progenitors but also as a critical enabler for viral oncogenesis.

A Non-Coding RNA Network Involved in KSHV Tumorigenesis.

Regulatory pathways involving non-coding RNAs (ncRNAs), such as microRNAs (miRNAs) and long non-coding RNAs (lncRNA), have gained great relevance due to their role in the control of gene expression modulation. Using RNA sequencing of KSHV Bac36 transfected mouse endothelial cells (mECK36) and tumors, we have analyzed the host and viral transcriptome to uncover the role lncRNA-miRNA-mRNA driven networks in KSHV tumorigenesis. The integration of the differentially expressed ncRNAs, with an exhaustive computational analysis of their experimentally supported targets, led us to dissect complex networks integrated by the cancer-related lncRNAs Malat1, Neat1, H19, Meg3, and their associated miRNA-target pairs. These networks would modulate pathways related to KSHV pathogenesis, such as viral carcinogenesis, p53 signaling, RNA surveillance, and cell cycle control. Finally, the ncRNA-mRNA analysis allowed us to develop signatures that can be used to an appropriate identification of druggable gene or networks defining relevant AIDS-KS therapeutic targets.

Kaposi's Sarcoma-Associated Herpesvirus-Encoded Viral IL-6 (vIL-6) Enhances Immunoglobulin Class-Switch Recombination.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic gamma-herpesvirus that causes AIDS-associated Kaposi sarcoma (KS) and several lymphoproliferative disorders. During the humoral immune response antigen-activated mature B cells acquire functional diversification by immunoglobulin heavy chain (IgH) class-switch recombination (CSR). CSR is initiated by activation-induced cytidine deaminase (AID) which targets highly repetitive switch (S)-regions to mediate DNA double-stranded breaks (DSBs) in the IgH locus facilitating intramolecular recombination. Here we show that in the context of cytokine stimulation, CSR is enhanced in murine B cells exposed only to replication-competent KSHV in an environment of KSHV infection, which coincided with elevated AID transcripts. Using murine splenic B cells and the mouse lymphoma CH12F3-2 CSR system, we identified that vIL-6, but not murine IL-6, increased class-switching, which correlated with upregulated AID expression. Together, these data suggest a regulatory role for KSHV vIL-6 in functionally modulating B cell biology by promoting CSR, which may in part explain how KSHV infection influences humoral immunity and affect KSHV pathogenesis.