Microenvironment in Lymph Nodes Inhibits Apoptosis in CLL Cells

[Guest guest]

[1443] Lymphoid Microenvironment Inhibits Apoptosis in B-CLL Cells:

Involvement of PI3-K/AKT Pathway and PTEN. Session Type: Poster

Session, Board #571-I

Medhat Shehata, ne Schnabl, Dita Demirtas, f D.

Schwarzmeier, Hilgarth, Markus Duechler, Gaiger,

Rainer Hubmann, Ulrich Jaeger Clinic of Internal Medicine I,

Hematology and Hemostaseology, Medical University of Vienna, Vienna,

Austria; Clinic of Internal Medicine, Hematology and Hemostaseology,

K. Landsteiner Institute for Cytokine and Leukemia Research, Vienna,

Austria

The activation of anti-apoptotic mechanisms in the leukemic B cells

in chronic lymphocytic leukemia (B-CLL) through the interaction with

their microenvironment may lead to prolonged survival and the

accumulation of the malignant clone. The aim of this study is to

elucidate the influence of the lymphoid microenvironment in the

activation of the potent anti-apoptotic PI3-K/Akt pathway and to

investigate the pattern of expression of the tumor suppressor PTEN

in B-CLL.

Stromal fibroblasts of bone marrow (BMF), spleen (SF) and lymph

gland (LGF) were used as an in vitro model for lymphoid

microenvironment. Pharmacological inhibitors and siRNAs against PI3-

K and Akt were applied to explore the anti-apoptotic effect of this

pathway in B-CLL.

The results showed that co-cultivation of B-CLL cells with human

BMF, LGF, and SF significantly inhibited apoptosis and prolonged

survival of the leukemic cells in comparison to suspension cultures.

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 and Akt1. These inhibitors significantly reduced the

supportive effect of stromal fibroblasts and induced apoptosis in B-

CLL cells.

The leukemic cells were more sensitive to PI3-K inhibition than T

cells, monocytes and stromal fibroblasts. Induction of apoptosis was

associated with a significant decrease in the intracellular levels

of PIP3, PI3-K, PDK1, Akt1, NF-kappa B, IKK, and dephosphorylation

(activation) of PTEN.

Since PTEN activity, as a negative regulator for PI3-K signalling,

is controlled by its phosphorylation at the tail domain, we studied

the pattern of PTEN protein expression in B-CLL. Western blotting

demonstrated that the total PTEN in PBMC of B-CLL patients (n=40) is

comparable to healthy individuals (n=8). However, using

phosphospecific anti-PTEN antibody demonstrated that samples of B-

CLL patients highly express phosphorylated (inactive) forms of PTEN

in comparison to healthy persons.

In conclusion, the results demonstrate that 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 may

lead to the activation of this pathway. The data also suggest that

targeting this pathway represents a feasible therapeutic approach in

B-CLL.