Flavopiridol Decreases Mcl-1 and Initiates Early Mitochondrial Damage in Chronic Lymphocytic Leukemia (CLL) Cells.

We have noted impressive activity of the cyclin-dependent kinase inhibitor flavopiridol in advanced-stage CLL patients, including those with a deletion of chromosome 17p13. Using a novel, effective schedule of flavopiridol, substantial and sometimes dramatic evidence of tumor cell death is observed as early as 4–6 hours. This is accompanied by hyperkalemia, hyperphosphatemia, hypocalcemia, and dramatic elevation in LDH consistent with acute tumor lysis syndrome. Studies by several groups including our own have demonstrated that Mcl-1 protein and mRNA is down-regulated with flavopiridol. Mcl-1 is an important protein that contributes to mitochondrial membrane stability. We have therefore sought to determine the role of mitochondria disruption in the mechanism of action of flavopiridol. By flow cytometry using the voltage-sensitive dye JC-1, loss of mitochondrial membrane potential was detected in flavopiridol-treated whole blood as early as five hours, prior to the onset of annexin-V or propidium iodide staining. This is in contrast to in vitro studies using human serum, in which mitochondrial depolarization and annexin-V staining occurred simultaneously. In isolated CLL cells treated with flavopiridol in vitro, loss of mitochondrial membrane potential was not affected by inhibitors of caspases 8 or 9 or by the broad caspase inhibitor Z-VAD-fmk, although apoptosis was effectively blocked by Z-VAD-fmk and caspase-8 inhibitor, and to a lesser extent, caspase-9 inhibitor. Flavopiridol was also able to effectively induce apoptosis and mitochondrial membrane depolarization in Jurkat cell lines deficient in caspase-8 or its adapter protein FADD. Additionally, lymphoid cells overexpressing Bcl-2 are resistant to flavopiridol-mediated apoptosis relative to the vector control. This suggests there is not direct binding of flavopiridol or its metabolites to APAF-1 (cytosolic adapter protein) and apoptosome assembly, as this process is insensitive to Bcl-2 family proteins. Further mechanistic studies were undertaken using isolated liver mitochondria. While the electron transport system was not uncoupled in this system, potential mechanisms of mitochondrial injury in leukemic cells from CLL patients are currently under exploration. Taken together, these observations suggest that mitochondrial perturbation contributes significantly to the death process induced by flavopiridol. Further studies to identify the mechanism of mitochondrial perturbation will be essential to understanding flavopiridol’s mechanism of action and for predicting patients at risk for acute tumor lysis syndrome. (Support for this work was provided by the Samuel Waxman Foundation and the Leukemia & Lymphoma Society.)