The mTOR Inhibitor Temsirolimus (CCI-779) Induces Cell Cycle Arrest and Autophagy in Hodgkin Lymphoma (HL) Cell Lines and Enhances the Effect of the PI3-Kinase Inhibitor LY294002.

Abstract The PI3K/AKT/mTOR pathway is frequently activated in a variety of cancers giving hope that that this pathway may prove to be a good target for cancer therapy. We and others have recently reported that primary and cultured Hodgkin and Reed-Sternberg cells of HL express the active phosphorylated form of Akt (pAkt) which contribute to their survival. Furthermore, we have demonstrated that inhibition of PI3K by the small molecule LY294002 or Akt inhibition induced cell cycle arrest and apoptosis. However, at the present time there is no PI3K or Akt inhibitors in clinical trials. Instead, several inhibitors of the downstream mTOR kinase are already being evaluated in patients with cancer. Temsirolimus (CCI-779) is an mTOR inhibitor that demonstrated promising clinical activity in patients with relapsed mantle cell lymphoma. To explore the potential clinical activity of Temsirolimus in HL, we examined its efficacy in HL-derived cell lines (HD-LM2, L-428, and KM-H2). Here, we demonstrate that CCI-779 inhibited mTOR, as evident by the inhibition of ribosomal S6 phosphorylation as early as 6 hours, with a dose as low as 1 nM. Temsirolimus induced antiproliferative cytostatic effect in the same dose (1nM) in all HL cell lines, and this effect did not improve with higher concentrations (up to 1000 nM). Furthermore, the effect was predominantly cytostatic with little or no induction of apoptosis. This antiproliferative effect was predominantly due to cell cycle arrest in G0/G1 phase, and was associated with upregulation of p27 cell cycle protein. Although temsirolimus did not induce apoptosis, it induced autophagy, as determined by Acridine Orange staining of the acidic autophagic intracellular organelles and FACS analysis. Combination of 1nM CCI-779 with increasing concentrations of the PI3K inhibitor LY294002 showed a profound decrease in the IC50 of LY294002 in all 3 cell lines. Moreover, the combination of CCI-779 and LY294002 blocked cell cycle at G0/G1 phase more efficiently and significantly increased autophagic cells when compared with each agent alone, but failed to induce apoptosis. This effect was mediated in part by inhibiting a negative feedback loop involving AKT phosphorylation. Collectively, our data suggest that temsirolimus may have a therapeutic value in HL.