Lenalidomide up-regulates DKK1 in myeloma plasma cells through JNK. Microarray analysis of primary myeloma plasma cell samples showed up-regulation of DKK1 expression after 48 hours of in vivo exposure to lenalidomide (A) or thalidomide (B) used as single agents. ⊡ represents pretreatment values; ▪, posttreatment results. DKK1 mRNA level in each sample before and after treatment is expressed as Affymetrix signal (1000-unit increments). (C) JNK activation was evaluated by Western blot analysis of phosphorylated isoforms p46 JNK1 (p-p46) and p54 JNK2 (p-p54) in 4 of the 24 myeloma plasma cell samples shown in panel A. Number 1 represents samples before lenalidomide exposure; number 2, samples after lenalidomide exposure. (D) c-Jun activity was evaluated in nuclear extracts of the same 4 samples shown in panel C and expressed as relative value compared to each control. ⊡ represents samples before lenalidomide exposure; ▪, samples after lenalidomide exposure. (E) DKK1 relative expression was evaluated by real-time PCR in another 26 myeloma plasma cell samples after 24 hours of in vitro treatment with lenalidomide (100 μM) and compared to each sample with no treatment. DKK1 mRNA level in each sample before (⊡) and after (▪) lenalidomide is expressed as relative value using the ΔΔCt approach. (F) The effect of pretreating cells with the JNK inhibitor SP600125 (10 μM) on lenalidomide-induced up-regulation of DKK1 expression was evaluated by real-time PCR (expressed as relative value). Violet bars represent control samples; red bars, samples after lenalidomide treatment; green bars, samples treated with lenalidomide in the presence of the JNK-specific inhibitor SP600125; and blue bars, samples treated with the JNK inhibitor alone. (G) The inhibition of JNK activation induced by SP600125 was confirmed in all 12 samples shown in panel F by analyzing c-Jun–binding activity expressed in relative units. In panels D-G each sample is expressed as the mean ± SEM of 2 duplicates.