Leflunomide synergizes with lenalidomide in inhibition of MM cell growth in vitro and in vivo, at least in part through synergistic c-Myc inhibition. (A) Proteasome inhibitor MG132 (1 µM) reverses Ter-induced c-Myc inhibition. MM.1S, NCI-H929, and RPMI-8226 cells were treated with 200 µM Ter for 7 hours. MG132 was added during the last 4 hours. Quantification of c-Myc expression after normalization to Actin expression is shown on the right. (B) Len (20 µM), but not Ter (200 µM), inhibits expression of IRF4 protein in RPMI-8226, MM.1S, and NCI-H929 MM cells treated for 24 hours (left). Len (20 µM), but not Ter (200 µM), inhibits expression of Ikaros transcription factor family members in MM cells NCI-H929 and MM.1S treated for 24 hours (right). (C) Lef synergizes with Len in inhibition of c-Myc protein expression in MM.1S, RPMI-8226, and NCI-H929 cells. Cells were incubated for 48 hours with 100 µM Ter and/or 20 µM Len, as indicated, and c-Myc expression was monitored by western blot. (Right) Quantification of c-Myc expression after normalization to Actin expression. (D) Len synergizes with (left) Ter and (right) PIM447 in in vitro growth inhibition of MM.1S and NCI-H929 MM cells. Cells were treated with constant ratios of Len:Ter or Len:PIM447 for 72 hours, as indicated. CI values are presented. (E) Lef synergizes with Len in survival of MM.1S xenograft NSG mice. A total of 5 × 10⁶ MM.1S-Luc cells were IV injected; treatment (7-8 mice per group) was initiated 2 weeks after injection. Survival was used as the end point. (F) Representative bioluminescence images (left) and quantification of tumor size (right) of control-, Len-, Lef-, and Len+Lef–treated animals as described in panel E. CI, combination index; DMSO, dimethyl sulfoxide; ED, effective dose.