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

Background and objectives. Loss of response to TGF-b is thought to contribute to B cell chronic lymphocytic leukemia (BCLL) progression. Recent findings of over-activation of the TGF-b signal in FKBP12-knockout mouse prompted us to investigate if FK506, the canonical ligand of FKBP, can activate the TGF-b signal in BCLL. Design and Methods. We studied 62 BCLL samples with RAI/Binet stage of 0 to 4. The TGF-b signal was investigated by Western blot and flow cytometry. The levels of Bcl2-family members and death-associated-protein (DAP) kinase were also investigated by Western blot, whereas apoptosis was studied in flow cytometry. Downmodulation of FKBP12 was obtained by gene silencing with short interfering RNA. Results. Twenty-two out of 62 BCLL samples were sensitive to TGF-b-induced apoptosis. All but two of the responder samples underwent apoptosis also when cultured with FK506, but not with cyclosporine. Thirteen samples non sensitive to TGF-b 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 Bim and Bmf increased. A loss of mitochondrial membrane potential preceded caspase activation and cell death. Our study shows that FK506 removed FKBP12 from its binding to the TGF-b-receptor. FKBP12 release activated the receptor-kinase activity as suggested by the enhanced levels of phospho-Smad found in cells depleted of FKBP12. Interpretation and Conclusions. Our study shows that most BCLL cells escape the homeostatic control of TGF-b and that FK506 restores the TGF-b signal in a proportion of non responder samples. We demonstrate that FK506 activates the TGF-b receptor I kinase activity in BCLL which transduces apoptosis by a mitochondrial-dependent pathway.