The effect of FK506 on transforming growth factor ß signaling and apoptosis in chronic lymphocytic leukemia B cells

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Haematologica, Vol 93, Issue 7, 1039-1048 doi:10.3324/haematol.12402

The effect of FK506 on transforming growth factor ? signaling and apoptosis in

chronic lymphocytic leukemia B cells

Simona Romano1, Mallardo1, Federico Chiurazzi1, Rita Bisogni1,

D'Angelillo1, Raffaele Liuzzi2, Giovanna Compare1, Fiammetta Romano1

1 Department of Biochemistry and Medical Biotechnologies, Federico II

University, Naples

2 Institute of Biostructure and Bio-Imaging-National Research Council (CNR),

Naples, Italy

Correspondence: Fiammetta Romano, MD, Department of Biochemistry and

Medical Biotechnologies, Federico II University, via Pansini, 5. 80131. Naples,

Italy. E-mail:romano@...

Background: Loss of response to transforming growth factor-beta (TGF-? ) is

thought to contribute to the progression of chronic lymphocytic leukemia. Recent

findings of over-activation of the TGF-? signal in FKBP12-knockout mouse

prompted us to investigate whether FK506, the canonical ligand of FKBP, can

activate the TGF-? signal in chronic lymphocytic leukemia.

Design and Methods: We studied 62 chronic lymphocytic leukemia samples from

patients with Rai/Binet stage 0 to 4 disease. The TGF-? signal was investigated

by western blotting and flow cytometry. The levels of Bcl2-family members and

death-associated-protein kinase were also investigated by western blotting,

whereas apoptosis was studied in flow cytometry. Down-modulation of FKBP12 was

obtained by gene silencing with short interfering RNA.

Results: Twenty-two out of 62 chronic lymphocytic leukemia samples were

sensitive to TGF-?-induced apoptosis. All but two of the responsive samples

underwent apoptosis also when cultured with FK506, but not with cyclosporine.

Thirteen samples that were not sensitive to TGF-? were sensitive to FK506.

Overall, response to FK506 occurred in 33 samples. FK506 induced Smad2

phosphorylation and nuclear translocation. Accordingly, death-associated-protein

kinase, a transcriptional target of Smad, was induced. At the same time, Bcl-2

and Bcl-xL levels decreased whereas the levels of Bim and Bmf increased. A loss

of mitochondrial membrane potential preceded caspase activation and cell death.

FK506 removed FKBP12 from its binding to the TGF-?-receptor. FKBP12 release

activated the receptor-kinase activity as suggested by the enhanced levels of

phospho-Smad found in cells depleted of FKBP12.

Conclusions: Our study shows that most chronic lymphocytic leukemia cells escape

the homeostatic control of TGF-? and that FK506 restores the TGF-? signal in a

proportion of non-responsive samples. We demonstrated that FK506 activates TGF-?

receptor I kinase activity in chronic lymphocytic leukemia, which transduces

apoptosis by a mitochondrial-dependent pathway.