Inhibition of apoptosis and long survival leads to accumulation of the leukemic cells in B cell chronic lymphocytic leukemia (B-CLL). This could be due to activation of anti-apoptotic cascades in CLL cells through interaction with their lymphoid microenvironment. Therefore, we investigated the role of tumor microenvironment in prolongation of survival of B-CLL cells and activation of the potent anti-apoptotic PI3-K/Akt pathway.
Stromal fibroblasts of bone marrow (BMFs), spleen (SF) and lymph gland (LGF) were used as an in vitro model for lymphoid microenvironment and we tested their ability to inhibit spontaneous apoptosis of B-CLL cells. Co-culture of B-CLL cells with human BMFs, LGF, and SF significantly inhibited apoptosis and prolonged survival of the leukemic cells in comparison to suspension cultures and to co-cultures with fibroblasts obtained from non-lymphoid organs. Trans-well culture experiments indicated that cell-cell interaction and soluble mediators are essential for this supportive effect. To explore the involvement of PI3-K/Akt pathway in the anti-apoptotic effect of stromal fibroblasts, co-cultures were performed in presence of PI3-K inhibitors (wortmannin or Ly294002) or siRNAs against PI3-K (p110ß subunit) and Akt1. These inhibitors significantly reduced the supportive effect of stromal fibroblasts and induced apoptosis in B-CLL cells. Interestingly, the leukemic cells were far more sensitive to PI3-K inhibition than T cells, monocytes and fibroblasts. Induction of apoptosis was associated with a significant decrease in the intracellular PIP3, PI3-K, PDK1 and Akt1, NF-kappa B, IKK and de-phosphorylation/activation of tumor suppressor protein PTEN. Studies using phosphospecific anti-PTEN antibody demonstrated that PBMC of CLL patients (n=40) highly express a phosphorylated form of PTEN. The results demonstrate that the PI3-K/Akt pathway is involved in inhibition of apoptosis of B-CLL cells and suggest that interaction of the leukemic cells with lymphoid microenvironment maintains the activation of this pathway. The data also suggest that targeting this pathway represents a new option for designing novel therapeutic strategies in B-CLL.