The Glycogensynthase Kinase 3b (GSK-3β) Is Involved in Leukemic Transformation

Purpose: Signal transduction pathways, such as the PI3K/AKT cascade, are frequently activated in acute myeloid leukemia and stimulate the proliferation and survival of leukemic cells. Mutations in upstream genes such as Class III receptor tyrosine kinases are frequent but not exclusive causes of that activation. An important downstream target of PI3K/AKT is the key regulator GSK-3β. It controls anti-apoptotic genes such as NF-kappaB and Cyclin D1, is involved in wnt-pathway activation and drug resistance of leukemic cells. Deregulated signaling by GSK-3β occurs through inhibition by AKT-mediated phosphorylation. We have observed that constitutive phosphorylation of GSK-3β occurred in hematopoietic cells with pro-leukemogenic PI3K-mutations. We wanted to evaluate the relevance of GSK-3β inactivation in the transformation process of hematopoietic cells.

Methods and Results: We used an in vitro factor-independent growth assay, GSK-3b inhibitors (Lithium, BIO) and established a second hit model using retroviral gene transfer of the weak oncogene Bcl X_L. Signaling cascades were analysed by western blot. We demonstrate that inactivation of GSK-3b alone was not sufficient to induce factor-independent growth in IL-3 dependent early hematopoietic cells (Ba/F3). Induction of apoptosis upon growth factor withdrawal was reduced, but not prevented, in the presence of GSK-3b inhibitors, leading to a delayed Caspase 3 activation, PARP cleavage and DNA fragmentation. Overexpression of Bcl-X_L also did not result in a prevention of apoptosis. GSK-3b inhibition in synergy with Bcl-X_L overexpression resulted in the establishement of several growth factor-independent cell lines, which were characterized by the activation of multiple signaling cascades including AKT, MAPK, STAT5, but not STAT3. Also Cyclin D1 was overexpressed in contrast to other cyclines (D2, D3) which are no substrates of GSK-3b.

Conclusions: Our data show that GSK-3β is part of the apoptotic response to growth factor withdrawal and suggest that GSK-3β is causaly involved in the transformation process of hematopoietic cells and seems to have an synergistic role in addition to other pro-leukemic mutations.