Abstract 939: Development of a dual fluorescent reactivation reporter Epstein-Barr virus (DFRR-EBV) to optimize lytic-induction therapy of EBV-associated cancers

Abstract Epstein-Barr virus (EBV) is a highly successful γ-herpesvirus, infecting >95% of the human population. Approximately 0.01% of infected individuals will develop an EBV-associated cancer later in life, resulting in 200, 000 annual cases globally. EBV-associated cancers, such as Diffuse Large B-Cell Lymphoma, are dependent on the expression of EBV latency genes for their continued growth and survival, making the disruption of latent infection a promising therapeutic target. “Lytic induction therapy” combines small molecules capable of reactivating EBV from latency with anti-viral drugs to increase toxicity to tumor cells and prevent EBV particle production. The full potential of this approach will require the development of second-generation lytic induction agents that induce a much greater proportion of tumor cells to enter the lytic cycle. Efforts to study the mechanisms of EBV reactivation are essential to this endeavor. EBV entry into the lytic phase is controlled by two viral transcription factors, Rta and Zta, and activators of the Rta and Zta promoters have previously been identified using reporter assays. These approaches, however, failed to capture major regulatory mechanisms that govern viral reactivation in EBV-positive tumors, such as long-range enhancer effects and epigenetic regulation of viral transcripts. To overcome these limitations, we constructed a dual fluorescent reactivation reporter virus (DFRR-EBV) that replaces Rta and Zta with GFP and RFP, respectively. This reporter is being used to screen a library of FDA-approved small-molecules to identify agents that reactivate EBV, in addition to a genome-wide unbiased CRISPR/Cas9 screen to identify negative regulators. Significantly, our reporter will distinguish between which small molecules are Rta versus Zta induction agents. Although Zta had been considered the principal regulator of the latent/lytic switch, we and others have demonstrated that expression of both Rta and Zta are essential for EBV reactivation1, 2. Therefore, the combination of an Rta induction agent with a Zta inducer should act synergistically, offering a significant improvement over the current approach of single-agent lytic induction therapies for EBV-associated cancers. References:1.Ali, A. et al. Rta is the principal activator of Epstein-Barr virus epithelial lytic transcription. PLoS Pathog 18, e1010886 (2022).2.Feederle, R. The Epstein-Barr virus lytic program is controlled by the co-operative functions of two transactivators. The EMBO Journal 19, 3080–3089 (2000). Citation Format: Emilie Greene, Eric Johannsen, Ahmed Ali. Development of a dual fluorescent reactivation reporter Epstein-Barr virus (DFRR-EBV) to optimize lytic-induction therapy of EBV-associated cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 939.