Previous studies showed that chronic lymphocytic leukemia (CLL) cells exhibit certain mitochondrial abnormalities including mtDNA mutations, increased superoxide generation, and aberrant mitochondrial biogenesis, which are associated with impaired apoptosis and reduced sensitivity to fludarabine. Here we report that CLL cells and multiple myeloma cells are highly sensitive to brefeldin A, an inhibitor of endoplasmic reticulum (ER) to Golgi protein transport currently being developed as a novel anticancer agent in a prodrug formulation. Of importance, brefeldin A effectively induced apoptosis in fludarabine-refractory CLL cells. Disruption of protein trafficking by brefeldin A caused the sequestration of the prosurvival factors APRIL and VEGF in the ER, leading to abnormal ER swelling and a decrease in VEGF secretion. Such ER stress and blockage of secretory protein traffic eventually resulted in Golgi collapse, activation of caspases, and cell death. Notably, the cellular sensitivity to this compound appeared to be independent of p53 status. Taken together, these findings suggest that malignant B cells may be highly dependent on ER-Golgi protein transport and that targeting this process may be a promising therapeutic strategy for B-cell malignancies, especially for those that respond poorly to conventional treatments. IntroductionChronic lymphocytic leukemia (CLL) is a common adult leukemia with unique characteristics in that the disease progression involves the persistent accumulation of apparently quiescent B cells, primarily due to defects in apoptosis rather than increased cell proliferation. 1 The apoptotic resistance of CLL cells has been attributed, in part, to high expression of antiapoptotic proteins such as Bcl-2, Bcl-X L , and Mcl-1. [2][3][4][5][6][7][8] Our previous work demonstrated that CLL cells contain multiple mitochondrial defects including mtDNA mutations, aberrant mitochondrial biogenesis, and increased generation of the oxygen radical superoxide, which is predominantly produced in the mitochondria. These abnormalities are associated with alterations in sensitivity to anticancer agents, suggesting that they may compromise the execution of the mitochondrial apoptotic machinery. [9][10][11] Fludarabine and alkylating agents have significantly improved clinical outcomes of CLL treatment. However, there are currently limited effective therapeutic options for patients that are refractory to these agents. 1,12,13 Thus, identification and evaluation of novel agents for the treatment of refractory CLL are important and challenging tasks. While investigating the mechanistic basis of aberrant mitochondrial biogenesis activity in primary CLL cells, we observed that in addition to increased mitochondrial content, CLL cells also appear to have a more extensive endoplasmic reticulum (ER) network than normal B lymphocytes. 10 The abundance of ER membranes in CLL cells has also been noted in earlier investigations and suggests that ER function may be very important for their survival. Consequently, this or...

Proteasome inhibitors are potent inducers of apoptosis in isolated lymphocytes from patients with chronic lymphocytic leukemia (CLL). However, the reversible proteasome inhibitor bortezomib (PS-341; Velcade) did not display substantial antitumor activity in CLL patients. Here, we compared the effects of bortezomib and a new irreversible proteasome inhibitor (NPI-0052) on 20S chymotryptic proteasome activity and apoptosis in isolated CLL cells in vitro. Although their steady-state (3 hours) IC 50 s as proteasome inhibitors were similar, NPI-0052 exerted its effects more rapidly than bortezomib, and drug washout experiments showed that short exposures to NPI-0052 resulted in sustained (z24 hours) 20S proteasome inhibition, whereas 20S activity recovered in cells exposed to even 10-fold higher concentrations of bortezomib. Thus, brief (15 minutes) pulses of NPI-0052 were sufficient to induce substantial apoptosis in CLL cells, whereas longer exposure times (z8 hours) were required for commitment to apoptosis in cells exposed to equivalent concentrations of bortezomib. Commitment to apoptosis seemed to be related to caspase-4 activation, in that cells exposed to bortezomib or NPI-0052 could be saved from death by addition of a selective caspase-4 inhibitor up to 8 hours after drug exposure. Our results show that NPI-0052 is a more effective proapoptotic agent than bortezomib in isolated CLL cells and suggest that the chemical properties of NPI-0052 might also make it an effective therapeutic agent in CLL patients.