Leflunomide directly inhibits PIM protein kinase activity and impairs c-Myc signaling. (A) A screening assay described by Anastassiadis et al.²³  was applied to test the inhibitory activity of 200 µM Ter on ∼600 known kinases. Kinases that were inhibited >70% at 200 µM Ter are shown. (B) PIM1-3 in vitro kinase activity assays using increasing concentrations of Ter. (C) In silico docking studies suggest that Ter may bind to the ATP-binding site of PIM kinases. Top, the docking pose of Ter at the PIM-3 ATP-binding site. Cyan ribbon, kinase backbone; black dots, hydrogen bonds between the drug molecule and protein. Bottom, 2-dimensional protein-ligand interaction diagram. Ter forms 2 hydrogen bonds with G105 and D189, together with a salt-bridge interaction with K69. (D) Overexpression of PIM proteins in MM.1S cells stabilizes or induces c-Myc protein expression and expression of PIM downstream proteins. (E) siRNA-mediated knockdown of PIM proteins is associated with inhibition of c-Myc protein expression. (F) Western blotting showing inhibition of c-Myc protein expression in MM cells treated for 48 hours with 50 to 200 µM Ter. (G) Gene set enrichment analysis graph of c-Myc upregulated genes²⁴  upon treatment of MM.1S and RPMI-8226 cells with 200 µM Ter or control for 48 hours revealed that c-Myc signaling was significantly impaired (negative enrichment score) upon Ter treatment in both cell lines and replicates. The Menssen Myc data set contains genes upregulated by Myc after transduction of human umbilical vein endothelial cell cells with a Myc-expressing adenovirus. One representative result is shown for each cell line. Supplemental Figure 3H shows similar results using a separate, independent Myc data set.²⁵  ES, enrichment score; FDR, false discovery rate; siRNA, small interfering RNA.