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Primary effusion lymphoma (PEL) is an aggressive B-cell malignancy without effective treatment, and caused by the infection of Kaposi’s sarcoma-associated herpesvirus (KSHV), predominantly in its latent form. Previously we showed that the SUMO2-interacting motif within the viral latency-associated nuclear antigen (LANASIM) is essential for establishment and maintenance of KSHV latency. Here, we developed a luciferase based live-cell reporter system to screen inhibitors selectively targeting the interaction between LANASIM and SUMO2. Cambogin, a bioactive natural product isolated from the Garcinia genus (a traditional herbal medicine used for cancer treatment), was obtained from the reporter system screening to efficiently inhibit the association of SUMO2 with LANASIM, in turn reducing the viral episome DNA copy number for establishment and maintenance of KSHV latent infection at a low concentration (nM). Importantly, Cambogin treatments not only specifically inhibited proliferation of KSHV-latently infected cells in vitro, but also induced regression of PEL tumors in a xenograft mouse model. This study has identified Cambogin as a novel therapeutic agent for treating PEL as well as eliminating persistent infection of oncogenic herpesvirus., Author summary Primary effusion lymphoma is a common AIDS-associated malignancy caused by infection with Kaposi’s sarcoma-associated herpesvirus (KSHV), and is currently absence of efficient and specific treatment. Natural product from herbal medicines is a major source of drug discovery for the treatment of a variety of diseases. In this study, the authors demonstrated that Cambogin, a polycyclic polyprenylated acylphloroglucinols (PPAPs) isolated from the branches of Garcinia esculenta (a tropical evergreen tree and traditional cancer treatment across Southern Asia), is a potent and effective inhibitor of KSHV-latently infected cells at a low concentration (nM) in vitro and in vivo, through targeting viral LANASIM-SUMO2 interaction.

Kaposi's sarcoma associated herpesvirus is tightly linked to multiple human malignancies including Kaposi's sarcoma (KS), Primary Effusion Lymphoma (PEL) and Multicentric Castleman's Disease (MCD). KSHV like other herpesviruses establishes life-long latency in the infected host by persisting as chromatin and tethering to host chromatin through the virally encoded protein Latency Associated Nuclear Antigen (LANA). LANA, a multifunctional protein, is capable of binding to a large number of cellular proteins responsible for transcriptional regulation of various cellular and viral pathways involved in blocking cell death and promoting cell proliferation. This leads to enhanced cell division and replication of the viral genome, which segregates faithfully in the dividing tumor cells. The mechanism of genome segregation is well known and the binding of LANA to nucleosomal proteins, throughout the cell cycle, suggests that these interactions play an important role in efficient segregation. Various biochemical methods have identified a large number of LANA binding proteins, including histone H2A/H2B, histone H1, MeCP2, DEK, CENP-F, NuMA, Bub1, HP-1, and Brd4. These nucleosomal proteins may have various functions in tethering of the viral genome during specific phases of the viral life cycle. Therefore, we performed a comprehensive analysis of their interaction with LANA using a number of different assays. We show that LANA binds to core nucleosomal histones and also associates with other host chromatin proteins including histone H1 and high mobility group proteins (HMGs). We used various biochemical assays including co-immunoprecipitation and in-vivo localization by split GFP and fluorescence resonance energy transfer (FRET) to demonstrate their association.

Epstein-Barr virus (EBV) is linked to a broad spectrum of B-cell malignancies. EBV nuclear antigen 3C (EBNA3C) is an encoded latent antigen required for growth transformation of primary human B-lymphocytes. Interferon regulatory factor 4 (IRF4) and 8 (IRF8) are transcription factors of the IRF family that regulate diverse functions in B cell development. IRF4 is an oncoprotein with anti-apoptotic properties and IRF8 functions as a regulator of apoptosis and tumor suppressor in many hematopoietic malignancies. We now demonstrate that EBNA3C can contribute to B-cell transformation by modulating the molecular interplay between cellular IRF4 and IRF8. We show that EBNA3C physically interacts with IRF4 and IRF8 with its N-terminal domain in vitro and forms a molecular complex in cells. We identified the Spi-1/B motif of IRF4 as critical for EBNA3C interaction. We also demonstrated that EBNA3C can stabilize IRF4, which leads to downregulation of IRF8 by enhancing its proteasome-mediated degradation. Further, si-RNA mediated knock-down of endogenous IRF4 results in a substantial reduction in proliferation of EBV-transformed lymphoblastoid cell lines (LCLs), as well as augmentation of DNA damage-induced apoptosis. IRF4 knockdown also showed reduced expression of its targeted downstream signalling proteins which include CDK6, Cyclin B1 and c-Myc all critical for cell proliferation. These studies provide novel insights into the contribution of EBNA3C to EBV-mediated B-cell transformation through regulation of IRF4 and IRF8 and add another molecular link to the mechanisms by which EBV dysregulates cellular activities, increasing the potential for therapeutic intervention against EBV-associated cancers., Author Summary Interferon regulatory factor (IRF) family members have different roles in context of pathogen response, signal transduction, cell proliferation and hematopoietic development. IRF4 and IRF8 are members of the IRF family and are critical mediators of B-cell development. Enhanced expression of IRF4 is often associated with multiple myeloma and adult T-cell lymphomas. Furthermore, IRF8 can function as a tumor suppressor in myeloid cancers. Epstein-Barr virus (EBV), one of the first characterized human tumor viruses is associated with several lymphoid malignancies. One of the essential antigens, EBV encoded nuclear antigen 3C (EBNA3C), plays a critical role in EBV-induced B-cell transformation. In our study, we now demonstrate that EBNA3C forms a molecular complex with IRF4 and IRF8 specifically through its N-terminal domain. We show that IRF4 is stabilized by EBNA3C, which resulted in downregulation of IRF8 through proteasome-mediated degradation and subsequent inhibition of its tumor suppressive activity. Moreover, si-RNA-mediated inhibition of IRF4 showed a substantial reduction in EBV transformed B-cell proliferation, and also enhanced their sensitivity to DNA-damage induced apoptosis. Therefore, our findings demonstrated that targeted disruption of EBNA3C-mediated differential regulation of IRF4 and IRF8 may have potential therapeutic value for treating EBV induced B-cell malignancies.