• A Comparison of SUMOylation in HK1 and BL41 Cell Lines

      Suarez, Persia; School of Medicine
      Nearly 96% of the population is infected with Epstein Barr virus (EBV), a gammaherpesvirus that results in a life-long infection. EBV lytically infects B lymphocytes and epithelial cells, and it establishes latency in B lymphocytes. Latent EBV infection often evades the host’s immune system; however, the presence of the EBV genome in certain cancers suggests that the virus is associated with approximately 200,000 new cases of cancer, specifically Burkitt’s lymphoma, Hodgkin’s lymphoma (HL), and nasopharyngeal carcinoma (NPC), each year. One cellular process commonly dysregulated in cancers, including EBV-positive lymphomas, is the post-translational modification of lysine residues by the Small Ubiquitin-like Modifier (SUMO), and SUMOylation inhibitors have been proposed to have potential anti-cancer properties. Our recent work focused on the small molecule inhibitor ML-792, which decreases global levels of SUMOylated proteins in EBV-positive and EBV-negative B lymphocytes. Similar experiments repeated with paired EBV-negative and EBV-positive nasopharyngeal cell line HK1 revealed that ML-792 only inhibited SUMOylation processes in the EBV-positive epithelial cells and not in their EBV-negative counterparts. We hypothesized that EBV may differentially modulate SUMOylation processes in epithelial cells when compared with B lymphocytes. This study aims to elaborate on the role of EBV on SUMOylation in epithelial cells. Paired primary B lymphocytes and epithelial cells were examined to determine the expression of the SUMO machinery. Results showed that EBV infection coincided with increased levels of SUMO-modified proteins and the SUMO-activating enzyme (SAE1 and SAE2), but not the SUMO-conjugating enzyme (Ubc9). Global levels of SUMOylated proteins increased in EBV-positive HK1 cells when compared with their EBV-negative counterparts. However, RNA and protein levels of the SUMO machinery varied greatly, which led us to ask if the confluence of the epithelial cells affected EBV-mediated changes in cells. Results demonstrated that RNA levels of the SUMO machinery significantly increased in sub-confluent EBV-positive HK1 cells, but these changes were not as apparent at the protein level. EBV-medicated changes in the SUMO machinery were more apparent at the protein level in confluent cells. To mimic a more physiological environment, EBV-negative and EBV-positive HK1 cells were also grown using a modified air-liquid interface method to model the human airway. Results showed that the presence of EBV corresponded with increased levels of the SUMO-activating enzyme and the SUMO-conjugating enzyme. Furthermore, the pattern of SUMOylated proteins changed in EBV-positive cells when compared with their EBV-negative counterparts. Taken together, our findings demonstrate that EBV does manipulate the SUMO machinery in epithelial cells, but not to the same extent as it does in lymphocytes. Therefore, additional studies are needed to better understand the effect of EBV on global levels of SUMOylated proteins in epithelial cells, which could identify if SUMOylation inhibitors have a therapeutic potential in the treatment of EBV-positive epithelial cancers.
    • Deciphering the Role of Sumoylation During EBV Replication

      Jenkins, Jessica L; School of Medicine
      Epstein Barr Virus, a gamma herpes virus, is the known causative agent in infectious mononucleosis and is highly ubiquitous in nature. Although primary infection typically yields no long term issues, viral latency is associated with lymphomas and epithelial cell carcinomas. We documented that the presence of LMP1, the principal EBV oncogene, dysregulates cellular sumoylation processes in lymphoma tissues, modulates innate immune response, and maintains viral latency. Sumoylation is a dynamic process were target proteins are modified with free small ubiquitin like modifier (SUMO) proteins. The SUMO modification is vital for cellular processes including: immune response, DNA damage repair sensing, cell cycle progression, resistance to apoptosis, and metastasis. Several cancers display dysregulation of the sumoylation process, making the SUMO machinery a sufficient target for anti-cancer therapies. Known sumoylation inhibitors include natural extracts and antibiotics. However, many of these agents are nonspecific and/or demonstrate adverse effects like allergic reactions with botanical extracts. This piqued our interest in investigating synthetically engineered compounds along with a well-known natural extract inhibitor, Ginkgolic Acid (GA). ML-792, 2-D08, and TAK-981 are synthetically derived small molecule inhibitors that were identified as selective SUMO-inhibitors, interfering at different stages of the sumoylation process. We hypothesize that the SUMO-inhibitors will have therapeutic effects for the treatment of EBV-associated malignancies by modulating the EBV life-cycle. Results showed that each of the tested inhibitors decreased global levels of sumoylated proteins, though ML-792 and TAK-981 showed greater inhibition when compared to GA and 2-D08. Additionally, the SUMO-inhibitors induced low levels of spontaneous reactivation in latently infected B cells. We also confirm that sumoylation is important for maintaining EBV latency and lytic replication in B cells. Lastly, we note anti-viral potential for each tested inhibitor, particularly GA and 2-D08 have a better affect than ML-792 and TAK-981 in this regard. Of the tested sumoylation inhibitors, we now propose 2-D08 as the best potential therapeutic drug to aid the treatment of EBV-associated malignancies due to its ability to significantly reduce viral DNA levels following induced reactivation and decrease the ability of produced virus to infect additional cells.