Rapamycin sensitizes tumor cells to chemotherapy-induced apoptosis and induces cell cycle arrest in ALK+ anaplastic large cell lymphoma.

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Anaplastic large cell lymphoma (ALCL) is a high grade type of non-Hodgkin lymphoma that frequently carries the t(2;5)(p23;q35) resulting in overexpression of nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK). Previous studies have shown that NPM-ALK activates the oncogenic phosphatidylinositol 3-kinase (PI3K)/AKT pathway. Activated (^Ser473phosphorylated) AKT phosphorylates the mammalian target of rapamycin (mTOR) protein that, in turn, phosphorylates two downstream effectors, p70S6K and ribosomal protein (rp)-S6, involved in protein translation. The effects of inhibition of mTOR signaling in ALK+ ALCL are unknown. Two ALK+ ALCL cell lines, Karpas 299 and SU-DHL1, were used. Cell viability was assessed 24 and 48 hours after incubation with increasing concentrations of rapamycin alone or rapamycin with doxorubicin, or rapamycin with the selective PI3K inhibitor, LY294002, using the trypan blue exclusion assay. Apoptotic cell death was assessed by immunofluorescence (DAPI staining), and flow cytometry (Annexin V binding) methods. S-phase fraction of the cell cycle was assessed using BrdU incorporation and ELISA methods. Expression levels of apoptosis- and cell cycle-related proteins were evaluated using Western blot analysis with specific antibodies. Treatment with rapamycin alone resulted in marked cell death at a concentration of 40 nM for Karpas 299 and 20 nM for SU-DHL1. Cell death in both cell lines was associated with apoptotic cell morphology observed as early as 16 hours following treatment, a significant increase in Annexin V binding, downregulation of the anti-apoptotic proteins Bcl-2, full length Mcl-1 and cFLIP, and cleavage of caspases 9 and 3. The combination of rapamycin with doxorubicin resulted in a further decrease in the viability of ALCL cells at lower concentrations for both agents. The combination of rapamycin with LY294002 also showed a synergistic effect on apoptotic cell death in ALCL cells. Moreover, treatment of Karpas 299 with rapamycin resulted in decreased S-phase fraction of cell cycle, which was associated with upregulation of cyclin-dependent kinase inhibitor p27 and increased levels of unphosphorylated RB. In addition, treatment of ALCL cells with rapamycin, alone or combined with either doxorubicin or LY294002, inhibited phosphorylation of mTOR and its downstream effectors p70S6K and rp-S6, and downregulated expression of total 4E-BP1 and eIF-4E proteins. We conclude that rapamycin induces cell cycle arrest and further sensitizes ALK+ ALCL cells to chemotherapy-induced apoptosis and these effects are mediated by modulation of cell cycle regulators and anti-apoptotic proteins. Inhibition of mTOR and its downstream effectors by rapamycin or currently used rapamycin analogs may have therapeutic implications in ALK+ ALCL.