Differential Effects of Three Small Molecules Blocking Phosphatidylinositol-3 Kinase or AKT in Hodgkin Disease Cell Lines: Induction of Apoptosis and Cell Cycle Arrest.

Several types of tumor cells utilize the phosphatidylinositol 3 Kinase (PI3K)/AKT pathway to support their growth and survival, and blockade of this pathway has been shown to have an antiproliferative effect in these cells. The significance of this survival pathway in Hodgkin disease (HD), and in particular, the neoplastic Hodgkin and Reed-Sternberg (H/RS) of Hodgkin disease (HD) is unknown. We studied routinely processed tissue sections of 42 cases of primary HD (n=42, 35 classical HD and 7 nodular lymphocyte predominant) for the expression of the active phosphorylated form of AKT. We also studied AKT expression in a panel of 4 well-characterized HD-derived cell lines (HD-MyZ, HD-LM2, L-428, and KM-H2). 27 of 42 (64.3%) cases of primary HD sections expressed phospho AKT (23/35 classical and 4/7 nodular lymphocyte predominant). Cultured H/RS cells also showed constitutive phosphorylation of AKT on Ser473. CD30 ligand (CD30L), CD40L and RANKL, increased the phosphorylation of AKT and downstream molecules as early as 1 hour. The effect of 3 small molecule inhibitors of PI3K/AKT pathway (PI3K inhibitor LY294002; AKT inhibitors QLT0394 and QLT0395 from QLT Inc, Vancouver, Canada) on cell proliferation and survival of cultured H/RS cells was examined by the MTS assay. The PI3K inhibitor LY294002 showed antiproliferative activity in a time and dose dependent manner in all cell lines tested. This antiproliferative effect was primarily due to cell cycle arrest in G0/G1 phase, as determined by propidium iodide staining, and to a less extent due to induction of apoptosis, as determined by the Annexin-V binding assay. However, when the AKT inhibitors QLT0394 and QLT0395 were used, they primarily induced apoptosis even at nanomolar doses. Apoptosis was mediated by caspase 8 activation as determined by western blot and was partially reversed by the pancaspase inhibitor ZVAD-FMK. Both AKT inhibitors alteredf AKT phosphorylation within 24 hours, and reduced phosphorylation of downstream molecules, such as 4E-BP1. Moreover, the AKT inhibitor QLT0395 showed downregulation of MDM2 and cyclin D1 within 24 hours, and increased ERK1/2 phosphorylation with subsequent decrease of total ERK1/2 levels at the same time frame, but had no effect in BCL-2, cFLIP or Bax. Importantly, both AKT inhibitors (QLT0394 and QLT0395) maintained their killing activity even in the presene of CD30L, CD40L, and RANKL. These preclinical data suggest that small molecules hitting different targets within PI3K/AKT pathway may have different effects on proliferation and apoptosis and that blockade of this pathway may be of therapeutic value in the treatment of patients with HD.