Zwernik SD, Adams BH, Yin J, Konduri SD, Rovin RA, Akhtar P. P53 level and MGMT promoter methylation are playing a role in Zika virus replication in glioblastoma. Poster presented at: Aurora Scientific Day; May 20, 2020; virtual webinar hosted in Milwaukee, WI.
Poster presented at: Aurora Scientific Day; May 20, 2020; virtual webinar hosted in Milwaukee, WI.
Background: Glioblastoma (GBM) is a malignant primary brain cancer. The poor median survival rate for patients with GBM of 15 months has not budged for the past 15 years, when the current standard treatment was first approved. There is no standard-of-care chemotherapy for recurrent GBM. Needless to say, novel treatments and treatment strategies for GBM are needed. One such novel treatment strategy is an oncolytic virus. The Zika virus’ (ZIKV) affinity for fetal neural stem cells has made it a compelling candidate as an oncolytic therapy for GBM. Previous studies have shown that ZIKV does infect and replicate in most but not all GBM cancer cells. Understanding the genetic milieu that is permissive for ZIKV infection is critical to the creation of a safe and effective viral oncolytic treatment. This report presents initial data for a ZIKV replication gene signature.
Purpose: We hypothesized that p53 level and MGMT promoter methylation are playing a role in ZIKV replication in GBM cancer cells.
Methods: ZIKV strain MR766 was propagated in Vero cells. Viral stock was titrated by plaque assays. Western blot was used to characterize MGMT, AXL, p53, and Sox2 expression in 8 commercial GBM cell lines (LN229, U87, A172, U251, LN18, T98G, U137, and U118). The cell lines were stratified by MGMT promoter methylation status and were exposed to ZIKV at a multiplicity of infection of 1. The percentage of infected cells was quantified by flow cytometry using the pan-flavivirus anti-E protein Ab 4G2. We also compared RNA-seq data of ZIKV replicating and nonreplicating GBM cell lines to find differentially expressed genes.
Results: In this study, we found that AXL was overexpressed in all 8 commercial GBM cell lines. With flow cytometry, we found that productive ZIKV replication only occurs in the MGMT-methylated (LN229, A172, U87, and U251) cell lines and not in MGMTunmethylated (T98G, LN18, U118, and U138) cell lines. qRT-PCR data showed that ZIKV enters all 8 cell lines but can only replicate in MGMT-methylated cell lines. Additionally, p53 expression trended lower in MGMT-methylated cell lines. RNA sequencing data comparing ZIKV replicating and nonreplicating GBM lines has identified multiple differentially genes.
Conclusion: Based on these results, there is a clear difference in the ability of the Zika virus to replicate in glioblastoma cell lines based on MGMT and p53 expression. Additional work is underway to understand the mechanism(s) underlying these findings and to define a ZIKV replication gene signature.