Role of NOXA and its ubiquitination in proteasome inhibitor-induced apoptosis in chronic lymphocytic leukemia cells.

BACKGROUND Bortezomib has been successfully used in the treatment of multiple myeloma and has been proposed as a potential treatment for chronic lymphocytic leukemia. In this study we investigated the mechanism by which bortezomib induces apoptosis in chronic lymphocytic leukemia cells. DESIGN AND METHODS Using western blot analysis, we monitored the regulation of BCL2 family members, proteins of the unfolded protein response (endoplasmic reticulum stress response) and activation of caspases in relation to induction of apoptosis (measured by annexin-propidium iodide staining and loss of mitochondrial membrane potential) by bortezomib in chronic lymphocytic leukemia cells. RESULTS Bortezomib induced apoptosis through activation of the mitochondrial pathway independently of changes associated with endoplasmic reticulum stress. Perturbation of mitochondria was regulated by a rapid and transcription-independent increase of NOXA protein, which preceded release of cytochrome c, HtrA2, Smac and activation of caspase-9 and -3. NOXA had a short half life (approximately 1-2 h) and was ubiquitinated on at least three primary lysine residues, resulting in proteasomal-dependent degradation. Down-regulation of NOXA, using short interfering RNA in chronic lymphocytic leukemia cells, decreased bortezomib-induced apoptosis. Finally bortezomib when combined with seliciclib resulted in a stronger and earlier increase in NOXA protein, caspase-3 cleavage and induction of apoptosis in chronic lymphocytic leukemia cells. CONCLUSIONS These results highlight a critical role for NOXA in bortezomib-induced apoptosis in chronic lymphocytic leukemia cells and suggest that this drug may become more efficient for the treatment of chronic lymphocytic leukemia if combined with other agents able to interfere with the basal levels of MCL1.