Altered expression of Bcl-2-family protein plays a central role in apoptosis dysregulation in cancer and leukemia, promoting malignant cell expansion and contributing to chemoresistance. Anti-apoptotic Bcl-2-family proteins Bcl-2 and Bcl-XL have been thoroughly validated as drug discovery targets for cancer, and strategies for inhibiting these proteins have been devised based on mimicking their endogenous antagonists, the BH3-only proteins. CLL (chronic lymphocytic leukemia) is a quintessential example of a human malignancy caused by defective programmed cell death, representing the most common form of adult leukemia in North America and Europe. Over-expression of the Bcl-2 protein is one of the most consistent and prominent etiological factors associated with this disease. In this study, we evaluated biological effects of ABT-737, a novel fully synthetic Bcl-2/Bcl-XL antagonist developed at Abbott Laboratories, on CLL cells and B-lymphoma cell lines in vitro. ABT-737 induced a striking dose-dependent apoptosis in all CLL cells tested, with a Lethal Dose 50% (LD50) of 3–10 nM for 10 of 11 CLL samples. ABT-737 exhibited similar activity against chemo-naive cells as well as extensively treated, relapsed-disease. In contrast, an enantiomer of ABT-737 with little affinity for Bcl-2 and Bcl-XL was inactive at concentrations up to 1 μM, demonstrating specificity. Similarly, ABT-737 induced striking dose-dependent apoptosis in B-lymphoma cell lines, such as 380 lymphoma cell line which over-expressed Bcl-2 as a result of t14:18 translocation, with the LD50 ratio for ABT-737:Enantiomer control of approximately 1,000. At concentrations up to 0.1 μM, ABT-737 had no cytotoxic action on normal T-lymphocytes isolated from peripheral blood collected from healthy individuals. In addition, CLL cells were at least 10 fold more sensitive to ABT-737 than normal CD19-positive B-lymphocytes isolated from healthy individuals. Moreover, ABT-737 induced caspase 3 activation and PARP cleavage within 2 hours in CLL B-cells, while zVAD-fmk completely blocked caspase 3 activation and PARP-cleavage. The mechanism of ABT-737 was further validated by confocal time-lapsed microscopy experiments, where the active compound (but not enantiomer control) was demonstrated to displace a Green Fluorescent Protein (GFP)-tagged BH3-containing protein from wild-type Bcl-XL localized at mitochondrial surfaces in intact tumor cell lines. In contrast, active compound failed to displace GFP-BH3 protein from the mitochondrial surface of cells expressing mutants of Bcl-XL purposely engineered to be incapable of binding ABT-737. Taken together, these data strongly suggest that ABT-737 is a mechanism-based inhibitor of Bcl-2 and Bcl-XL that warrants further evaluation for the possible treatment of CLL and other malignancies linked to over-expression of Bcl-2 or Bcl-XL, where chemorefractory states represent a barrier to successful eradication of cancer.

Special thanks to Dr. Rassenti, Ph.D., CRC Tissue Core Director, for CLL samples.

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