NPM-ALK phosphorylates MDM2 on serine 166. (A) MDM2 is phosphorylated on serine 166 in archival biopsy specimens isolated from NPM-ALK–expressing ALCL (top left panel). Conversely, ALK⁻ cases do not strongly express MDM2 in a phosphorylated form (top right panel). All cases were screened for the presence of a translocation involving ALK on chromosome 2 by FISH using an ALK break-apart probe (bottom left panel shows an ALK⁺ case) and for the expression of MDM2 by immunohistochemistry (bottom right panel shows a case representative of all samples regardless of ALK status). Representative samples are shown of the 10 ALK⁺ cases screened. Likewise MDM2 is phosphorylated on serine 166 in patient-derived ALK-expressing ALCL cell lines SUHL-1, Karpas-299, and DEL (B) and in BaF3 cells expressing NPM-ALK. Numbers represent arbitrary units after densitometric analysis and normalization for the tubulin loading control. (C) Treatment of SUDHL-1 cells with either (D) siRNA against ALK for 48 hours (compared with a scrambled sequence [Scr]) or (E) 10 μM of the Jak3/ALK inhibitor WHI-P154 for 2 hours, causes a reduction of pMDM2 levels in the cells. Cells were analyzed by trypan blue staining at the time of analysis to exclude significant cell death. Furthermore, 48 hours of treatment with a PI 3-kinase inhibitor (20 μM LY294002) attenuated MDM2 phosphorylation. Numbers represent arbitrary units after densitometric analysis and normalization to the loading control; vertical lines have been inserted to indicate a repositioned gel lane (F). The inhibitor compounds had no effect on MDM2 transcript levels as determined by qRT-PCR (G). Error bars represent SDs of the mean. Results are representative of at least 3 independent experiments.