High-throughput drug screen identifies pharmacologic agents that induce latency III antigen expression. (A) Immunoblot of BL cell lines to characterize latency. BC2, latency I control; LCL 9001, latency III control; Ramos, EBV⁻ control. (B) Heatmap showing the fold change in LMP1 for 2 replicates across 441 compounds. Dendrogram branches on the right illustrate groupings based on unsupervised clustering, highlighting a cluster of 33 compounds inducing a greater than twofold change in both replicates (blue branches). Inset shows the list of 33 compounds grouped based on similarity of pathway targets. (C) Network plot showing the pathway enrichments based on drug targets. Each node denotes a subpathway, with colors delineating pathway groupings (see table). Nodes with multiple colors denote shared pathway groupings. (D) Focused screen of epigenetic modifying agents. qRT-PCR for LMP1 and Cp promoter transcripts in cells treated with drug vs vehicle control for 48 hours. Data are shown as fold change in treated cells compared with vehicle control. Experiments were performed in duplicate. Drug doses were as follows: GSK-126, 5 μM; EPZ-6438, 5 μM; romidepsin, 0.25 nM; HDAC3i, 5 μM; panobinostat, 100 nM; 5-azacytidine, 4 μM; and decitabine, 1 μM. (E) Combination treatment with panobinostat and decitabine. qRT-PCR for LMP1 and Cp transcripts in cells treated with vehicle, panobinostat alone (100 nM), decitabine alone (1 μM), or combination. Experiments were performed in triplicate. Error bars represent standard error of the mean (SEM). DNMTi, DNA methyltransferase inhibitor; EZH2i, EZH2 inhibitor; FC, fold change; HDAC, histone deacetylase; HDACi, histone deacetylase inhibitor; LTR, long terminal repeat.