R-(−)-Gossypol (AT101) Binds to Bcl-2 Family Proteins and Induces Apoptosis in CLL.

Abstract

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of monoclonal B cells in the blood, secondary lymphoid tissues, and marrow. The leukemia cells primarily are arrested in the G0/G1 phase of the cell cycle and appear resistant to programmed cell death. Several anti-apoptotic proteins are over expressed in CLL and this correlates with resistance to treatment, disease progression and overall poor prognosis.Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L), Bcl-2, and Mcl-1, are overexpressed in many cancers including CLL and contribute to tumour initiation, progression and resistance to therapy. Mcl-1 is of particular interest because this molecule appears to be regulated by Nurse-like Cells and other stromal cells that promote survival of CLL cells in vitro and very likely also in vivo.These proteins enhance the resistance of CLL cells to spontaneous and/or drug-induced apoptosis primarily by interacting with, and antagonizing the activity of mitochondria membrane pro-apoptotic proteins such as Bax and Bak. The protein-protein interaction of Bcl-2 family members is critical for their activity, and these interactions are governed by binding to the BH3 domain. Racemic gossypol is found in cotton seeds and has been studied as a cytotoxic agent in cancer cell lines and in clinical studies in patients with a large variety of cancers. The antitumor activity of racemic gossypol appears to reside principally in the R-(−)-enantiomer (AT101), with reduced activity observed for the S-(+)-enantiomer. AT101 is an antagonist of the BH3-binding groove of the Bcl-2 family of proteins that can inhibit the interactions of these proteins with pro-apoptotic molecules.We examined whether AT101 could induce apoptosis in Chronic Lymphocytic Leukemia (CLL) and its ability to bind in vitro anti-apoptotic molecules from the Bcl-2 family. Using a Fluorescence Polarization Assay (FPA) we studied the competitive binding affinity of AT101 to Bcl-2 family member proteins. We observed that both, racemic gossypol and AT101 had comparable affinity for Bck-2, Bcl-B, Bfl-1 with EC50=0.6 to 10 μM range. AT101 had a stronger binding affinity to Bcl-X(L) (EC50=0.998 μM vs. 3.084 μM for racemic gossypol), and to Mcl-1 (EC50= 0.52μM vs. 1.07μM for racemic gossypol).CLL cells were incubated with AT101 for 48 hrs at different concentrations. We observed that leukemia cells were induced to undergo apoptosis in a time and dose dependent manner and that this effect was independent of ZAP-70 expression or IgVH gene mutational status (IC50= 2μM). Cells undergoing apoptosis showed PARP-1 cleavage and upregulation of pro-apoptotic molecules such as Bid, p53, as well as downregulation of Mcl-1.These results indicate that AT101 has stronger pan-specific binding affinity for Bcl-2 family proteins than racemic gossypol, in particular to Mcl-1 and Bcl-X(L), and that this compound induces apoptosis in CLL B cells independently of ZAP-70 expression or IgVH gene mutational status. Because of these encouraging results a clinical trial using AT101 in CLL patients with high-risk features is currently open at our institution.