Activation of alpha-diacylglycerol kinase is critical for the mitogenic properties of anaplastic lymphoma kinase

Oncogenic rearrangements of the tyrosine kinase receptor anaplastic lymphoma kinase (ALK), most commonly represented by the nucleophosmin/ALK fusion protein (NPM/ALK), are involved in the pathogenesis of anaplastic large-cell lymphomas (ALCLs). In an effort to identify new intracellular transducers operative in ALK-positive malignancies, we have investigated the potential involvement of diacylglycerol kinase (DGK). Here we show that DGK is constitutively activated in the NPM/ALK-positive ALCLderived cell line Karpas 299 and in NPM/ ALK-infected 32D hematopoietic cells. These results were further validated in fibroblastic NIH-3T3 cells expressing a previously described chimeric epidermal growth factor receptor (EGFR)/ALK molecule that allows dissection of ALK enzymatic function under conditions of controlled ligand-induced activation. In this cell system, we also show that ALKmediated DGK activation is dependent on p60src tyrosine kinase, with which DGK forms a complex. The specific inhibition of DGK, obtained by cell treatment with R59949, significantly reduced cellular growth in all cell lines. This result was further confirmed in Karpas 299 cells following specific down-regulation of DGK by RNA interference. Overall, our data indicate that DGK activation is involved in the control of ALK-mediated mitogenic properties.