EBNA1 Inhibitors Block Proliferation of Spontaneous Lymphoblastoid Cell Lines From Patients With Multiple Sclerosis and Healthy Controls

Abstract

Background and objectives: Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that establishes lifelong latency in memory B cells and has been identified as a major risk factor of multiple sclerosis (MS). B cell depletion therapies have disease-modifying benefit in MS. However, it is unclear whether this benefit is partly attributable to the elimination of EBV⁺ B cells. Currently, there are no EBV-specific antiviral therapies available for targeting EBV latent infection in MS and limited experimental models to study EBV in MS.

Methods: In this study, we describe the establishment of spontaneous lymphoblastoid cell lines (SLCLs) generated ex vivo with the endogenous EBV of patients with MS and controls and treated with either an Epstein-Barr virus nuclear antigen 1 (EBNA1) inhibitor (VK-1727) or cladribine, a nucleoside analog that eliminates B cells.

Results: We showed that a small molecule inhibitor of EBNA1, a critical regulator of the EBV life cycle, blocks the proliferation and metabolic activity of these SLCLs. In contrast to cladribine, a highly cytotoxic B cell depleting therapy currently used in MS, the EBNA1 inhibitor VK-1727 was cytostatic rather than cytotoxic and selective for EBV⁺ cells, while having no discernible effects on EBV^- cells. We validate that VK-1727 reduces EBNA1 DNA binding at known viral and cellular sites by ChIP-qPCR.

Discussion: This study shows that patient-derived SLCLs provide a useful tool for interrogating the role of EBV⁺ B cells in MS and suggests that a clinical trial testing the effect of EBNA1 inhibitors in MS may be warranted.

Figure 1. Detection of EBV and EBV Loads in Patients With MS and HCs

(A) Percent of HCs, SMS, and AMS PBMCs and CD19⁺ B cells that are positive for EBV by ddPCR (B) EBV-BAMH1 copies/10⁶ cells in PBMCs and CD19⁺ B cells from HCs, patients with SMS, and patients with AMS by ddPCR (0 on Y-axis represents a value that was below the limits of detection of the assay). EBV = Epstein-Barr virus; HCs = healthy controls; PBMC = peripheral blood mononuclear cell.

Figure 2. Generation of Spontaneous Lymphoblastoid Cell Lines (SLCLs) From the PBMCs of Patients With MS and Healthy Controls

(A) Patient with MS and control PBMC were used to generate SLCLs from endogenous EBV, without adding exogenous/laboratory strain EBV. After 21 days in culture, residual T-cells were eliminated by the addition of cyclosporin A. (B) Digital droplet PCR was performed on newly formed SLCLs. Digital droplet (dd) PCR plot indicates EBV-BamHI W-FAM (Y-axis; upper left quadrant; blue droplets) and the RPP30 cellular housekeeping gene-VIC (X-axis; lower right quadrant; green droplets). (C) Representative 2-dimensional ddPCR plot performed on a generated SLCL culture. Primer sets and fluorescent probes to detect a sequence in the EBV-BamHI W-FAM (Y-axis; upper left quadrant; blue droplets) and RPP30-VIC, housekeeping gene (X-axis; lower right quadrant; green droplets). Populations double positive for EBV-BamHI W and RPP30 are in the upper right quadrant (orange droplets). (D) Diagram of EBV genome. (E) Amplification of EBV WT DNA (≈1 Kb) with 3′ primer 1. (F) Amplification of B95.8 laboratory strain DNA (≈500bp) with 3′ primer 2. EBV = Epstein-Barr virus; PBMC = peripheral blood mononuclear cell.

Figure 3. Selective Inhibition of EBV⁺ SLCL Proliferation From Patients With MS and Controls by EBNA1 Inhibitor, VK-1727

(A) Structure of EBNA1 inhibitor, VK-1727. (B) Percent inhibition of proliferation as measured by [³H] Thymidine incorporation at day 7 in 2 EBV⁻ B cell lines (BJAB and Ramos) and 6 EBV⁺ SLCLs treated with VK-1727 (2–10 µM). (C) Cell counts (right Y-axis) and [³H] Thymidine incorporation (left Y-axis) in EBV⁻ B cell lines (BJAB and Ramos). (D) Cell counts (right Y-axis) and [³H] Thymidine incorporation (left Y-axis) EBV⁺ SLCLs from healthy controls and patients with MS. (C and D) Black bars indicate ³H-CPM when cells were seeded at 1 × 10⁴/well; gray bars indicate ³H-CPM when cells were seeded at 5 × 10³/well. Black and gray lines indicate the number of viable cells when cultures were seeded at 1 × 10⁴/well and 5 × 10³/well, respectively (*p =< 0.02–0.05; **p =< 0.006; ***p = 0.0006; ****p < 0.0001; Two-way ANOVA). EBV = Epstein-Barr virus; SLCL = spontaneous lymphoblastoid line.

Figure 4. Selective Inhibition of Cellular Metabolism by EBNA1 Inhibitor VK-1727 but Not Cladribine

(A) VK-1727 inhibition of cell metabolism in a resazurin-based assay using EBV⁺ SLCLs and EBV⁻ control (BJAB). Representative plots shown. (B) Cladribine inhibition of cell metabolism in a resazurin-based assay using EBV⁺ SLCLs and EBV⁻ control (BJAB). Representative plots shown. EBV = Epstein-Barr virus; SLCL = spontaneous lymphoblastoid line.

Figure 5. EBNA1 Inhibition Perturbs Cell Cycle Expression While Cladribine Induces Apoptosis in SLCLs

(A) Cell cycle profiles comparing EBV⁺ and EBV⁻ B cells treated with DMSO, VK-1727 (25 µM), or cladribine (2.5 µM) measured by flow cytometry analysis of propidium iodide staining (representative graphs for SMS1 and Ramos). (B) Graph of cell cycle kinetics data comparing the effects of VK-1727 with those of cladribine. EBV⁺ cells (HC1-2, SMS1-3, and AMS1-4) treated with 25 µM of VK-1727 show a significant decrease in the total population of G2 cells that is not observed for EBV⁻ (Ramos and BJAB) B cells. In addition, while cladribine induces apoptosis, marked by an increase in the <G1 population, in all B cells regardless of EBV infection, apoptosis was not increased in EBV⁻ or EBV⁺ cells treated with VK-1727. Each cell line was treated, stained, and analyzed as biological replicates. Data shown here are batched by group (EBV⁻, HC SLCL, SMS SLCL, and AMS SLCL) (****p < 0.0001, 2-way ANOVA). (C) Flow cytometry analysis of Annexin V/PI staining comparing EBV⁻ and EBV⁺ B cells with DMSO and VK-1727 (25 µM) or cladribine (2.5 µM) (representative graphs for SMS1 and Ramos). (D) Graphs summarizing live cell populations (% Q4) observed from the Annexin V/PI experiment. Each cell line was treated, stained, and analyzed as biological replicates. Data shown here are batched by group (EBV⁻, HC SLCL, SMS SLCL, and AMS SLCL) (****p < 0.0001, 2-way ANOVA). EBV = Epstein-Barr virus; SLCL = spontaneous lymphoblastoid line.

Figure 6. VK-1727 Disrupts EBNA1 Binding in SLCLs

(A) ChIP assay for EBNA1 binding to the DS, Qp, and cellular locus CLIC1 in AMS4 SLCLs treated with DMSO or 30 µM VK-1727 for 72 hours. p values were determined for 3 biological replicates (***p < 0.001, Two-way ANOVA). (B) Immunoprecipitation was performed with IgG as a control bottom panel. (C) Expression of EBV latency (EBNA1, EBNA, EBER, LMP1) and lytic (Zta and EA-D) genes in EBV⁺ AMS4 SLCL. p values were determined for 3 biological replicates (*p < 0.05, t test). EBV = Epstein-Barr virus; SLCL = spontaneous lymphoblastoid line.