PI3-K May be Drug Target in CLL

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IN VITRO MODEL FOR LYMPHOID MICROENVIRONMENT AND INHIBITION OF

APOPTOSIS IN B-CLL CELLS: INVOLVMENT OF PI3-K/AKT PATHWAY AND PTEN

0912

Author Shehata, Medhat , Vienna, Medical University of Vienna

Austria

Co-author(s) Schnabl, ne , Vienna, Medical University of Vienna

Demirtas, Dita , Vienna, Medical University of Vienna

Schwarzmeier, f , Vienna, KLI for Cytokines a. T.

Microenvironment

Hilgarth, , Vienna, Medical University of Vienna

Duechler, Markus , Vienna, Medical University of Vienna

Gaiger, , Vienna, Medical University of Vienna

Hubmann, Rainer , Vienna, Medical University of Vienna

Jaeger, Ulrich , Vienna, Medical University of Vienna

Background: B-CLL is considered as a disease of accumulation due to

the long survival and resistance of apoptosis of the leukemic B-

cells. Recent data suggest the presence of proliferation pool of B-

CLL cells in the lymphoid tissues and that 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. However, the signaling molecules, which are responsible for

these processes are not completely defined.

Aims: The aim of this study is to provide an in vitro model, which

mimics the in vivo situation in the lymphoid tissue, and to

elucidate the role of the lymphoid microenvironment in the

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

prolonging survival and maintenance of proliferation of B-CLL cells.

Method: Stromal fibroblasts in the lymphoid tissues are known to

produce several growth factors, cytokines and matrix proteins, which

are essential for supporting hematopoiesis. Therefore, bone marrow

(BMF), spleen (SF) and lymph gland (LGF) fibroblasts were used as an

in vitro model for lymphoid microenvironment under serum free

condition. Pharmacological inhibitors and siRNAs against PI3-K and

Akt were applied to explore the anti-apoptotic effect of the PI3-

K/Akt pathway in B-CLL.

Results: 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. The data also

demonstrated the presence of proliferation islands of B-CLL cells,

which were in close contact with the stromal cells. 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 the resting and

proliferating 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 PIP3, PI3-K, PDK1 and Akt1 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 lymphoid microenvironment model allows

demonstrating that the interaction between the leukemic cells and

the lymphoid microenvironment may lead to the activation of PI3-K

pathway and inhibition of apoptosis of B-CLL cells. The data also

demonstrate that the in vitro model may allow large-scale drug

screening and indicate that PI3-K is a feasible therapeutic target

in B-CLL.