Histone methylation and oncogenic signaling impact PDPN expression. (A) Irreversible blockade of EGFR phosphorylation by dacomitinib (PF) does not rescue PDPN expression in EGFRvIII-driven U373vIII cells (n = 3). (B) Inhibition of PI3K activity (downstream effector of EGFR) by pictilisib fails to restore PDPN expression in U373vIII cells (n = 3). (C) Treatment with 5-azacytidine (5-Aza) does not lead to reexpression of PDPN protein in U373vIII cells (n = 3). (D) Relationship between known EZH2-correlated genes and EZH2 (left panel) or PDPN (right panel) across GBM cells. In each GSEA plot, the x-axis shows the ranking of genes based on their correlation with EZH2 or PDPN across the single-cell GBM data set.³³  The vertical black lines denote the set of genes known to be positively correlated with EZH2, based on a meta-analysis of publicly available bulk tissue RNA-seq data sets.⁴⁰  (E) Treatment of U373vIII cells with the EZH2 inhibitor (UNC1999) leads to gradual and partial reexpression of PDPN over 25 days (25 D) (n = 5). (F) Enhancement of PDPN protein expression in U373vIII glioma cells cotreated with inhibitors of EZH2 (UNC1999) and EGFR (dacomitinib) suggests a role for histone H3 trimethylation and chromatin modification, as well as EGFR kinase signaling, in PDPN regulation (n = 3)