Adaphostin disrupts mitogenic and anti-apoptotic signaling by NPM-ALK in anaplastic large cell lymphomas

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Anaplastic Large Cell Lymphomas (ALCLs) characteristically have a chromosomal translocation t (2; 5), which results in a fusion protein with Anaplastic Lymphoma Kinase (ALK) and nucleophosmin (NPM). The constitutively active tyrosine kinase protein product is involved in mitogenic signaling through PLCδ1, as well as anti apoptotic signaling through PI3 Kinase, Stat5 and Stat3. Furthermore, NPM-ALK has been shown to physically associate with phosphorylated Src. The Developmental Therapeutics Program of the National Cancer Institute initially identified adaphostin as an inhibitor of p210 ^(Bcr/abl) kinase-positive K562 cells but subsequent studies indicated that it is also effective against p210 ^(Bcr/abl) kinase-negative cancer cells. However, the exact mechanism by which adaphostin inhibits various cancer cells is not yet completely understood. Here we show that the adaphostin (NSC 680410) inhibits proliferation and reduces viability of the NPM-ALK+ cell line, suDHL1, with an IC50 of 0.5uM over 24 hours. This treatment is also shown to reduce phosphorylation of NPM-ALK at tyrosine 664, which is required for signaling through PLCδ1, disrupt the physical interaction between NPM-ALK and PLCδ1, and partially inhibit Stat3 phosphorylation at tyrosine 705. Recently, in our laboratory, we have established that adaphostin inhibits some of the Src related kinases, and here we show that a concentration of 0.5uM adaphostin is sufficient to inhibit both Src phosphorylation at tyr 416 (required for its activation) and association between Src and NPM-ALK in suDHL1. Using cell-free in-vitro kinase assays we have also shown that adaphostin directly inhibits the activity of both Src and Alk at 1uM. These results highlight a role for adaphostin in the inhibition of mitogenic and anti apoptotic signaling pathways possibly regulated by NPM-ALK.