Stroma-Activated Integrin-Linked Kinase (ILK) Supports Survival of Leukemic Cells Via Stimulation of Notch-Hes Signaling: New Therapeutic Opportunities.

We have previously demonstrated that the BM microenvironment plays a crucial role in the pathogenesis of AML by influencing tumor growth, survival, and drug resistance. Integrin-linked kinase (ILK) has been shown to directly interact with β integrins and phosphorylate AKT in a PI3-kinase (PI3K)-dependent manner to promote cell survival and proliferation. HES-1 encodes a basic helix-loop-helix transcription factor downstream of the Notch receptor, and functions as a positive regulator of hematopoietic and neuronal stem cell self-renewal. Direct co-culture of human mesenchymal stem cell (MSC) and leukemic NB4 cells results in activation of PI3K/ILK/AKT signaling (elevated phospho(p)-Akt, p-GSK3β and nuclear-localized β-catenin), increased expression of Notch1 and Hes1 proteins and upregulation of p-STAT3 detected by Western blot and confocal microscopic analyses. Both, PI3K inhibitor LY294002 (20 μM) and ILK inhibitor QLT0254 (10 μM) specifically inhibited stroma-induced activation of AKT and Stat-3 signaling, suppressed GSK phosphorylation and decreased Notch 1 and HES1 expression. This resulted in massive induction of apoptosis which was not abrogated by stromal co-culture (AnnexinV positivity %, MSC(-) vs MSC(+); control 33.8±2.5 vs 27.3±1.9 p=0.02, QLT 51.4±2.5 vs 55.8±3.5 p=0.26, LY 47.0±8.1 vs 47.9±6.1 p=0.85, 48hrs). In contrast, GSK3b inhibitor BIO (0.1 μM) prevented the serum-withdrawal-induced apoptosis of NB4 cells (AnnexinV positivity %, control 38.1±4.0 vs BIO 25.9±3.4 p=0.003, 48hrs) with marked increase in Notch1 and Hes1 expression detected by confocal microscopy. These observations indicate that Notch signaling is involved in leukemic cell survival stimulated by BM stromal interactions via activation of the ILK-AKT-GSK3β pathway. We have next investigated the effects of leukemic cells on stroma cells. Coculture with NB4 cells caused significant increase in Hes1 and Bcl2 proteins in MSC along with phosphorylation of STAT3 and Akt, which were all abrogated by the treatment with QLT0254 or LY294002. In summary, these results demonstrate that interactions of leukemic and bone marrow stromal cells result in activation of PI3K/ILK/AKT and Notch-Hes signaling in both, leukemic and stromal cells. Disruption of these interactions by specific ILK inhibitors represents a novel therapeutic approach to eradicate leukemia in the bone marrow microenvironment via direct effects on leukemic cells and by targeting activated bone marrow stromal cells.