Abstract
Abstract 3510
The reversible proteasome inhibitor bortezomib is effective in the treatment of multiple myeloma and mantle cell lymphoma, but failed to produce objective responses in chronic lymphocytic leukemia (CLL). Carfilzomib (CFZ) is a tetrapeptide ketoepoxide that belongs to a new class of irreversible proteasome inhibitors that specifically target the chymotrypsin-like subunit of the 26S proteasome. Based on preclinical data demonstrating potent cytotoxicity in primary CLL cells, CFZ is currently in a phase I clinical trial at The Ohio State University in patients with relapsed or refractory CLL. However, the mechanism of action of CFZ in CLL is unknown. We have therefore investigated the effects of CFZ on CLL patient cells ex vivo.
Here we demonstrate that a short (1 hr) exposure of 100 nM CFZ is sufficient to inhibit the chymotrypsin-like proteasome subunit in CLL cells. This exposure is also rapidly cytotoxic, inducing apoptosis in approximately 50% of cells by 24 hr (annexin+ and/or PI+). Unlike bortezomib, the cytotoxicity of carfilzomib is not diminished in media with human serum compared to fetal bovine serum. Additionally, CFZ is more cytotoxic to normal CD19+ B cells than normal CD3+ T cells at clinically relevant concentrations of 33 to 300 nM, suggesting that this agent could potentially avoid immune-suppressing T-cell depletion that is commonly noted with chemotherapy. CFZ causes CLL cell death ex vivo by a caspase-dependent apoptotic pathway, indicated by PARP cleavage and rescue by the broad caspase inhibitor Boc-D-fmk. Importantly, our studies indicate that CFZ causes cytotoxicity in primary CLL cells irrespective of p53 status. This tumor suppressor, which is functional in most CLL patients at the time of diagnosis, is mutated or deleted in at least one allele in approximately 40% of patients with advanced CLL, and p53 pathway dysfunction is associated with resistance to standard therapies and poor overall survival. Therefore, the ability of CFZ to induce apoptosis irrespective of p53 function is of important clinical significance. Additionally, the pro-apoptotic protein Noxa is increased following CFZ treatment despite a lack of induction in mRNA, suggesting accumulation of protein via inhibition of proteasome-mediated degradation.
The NF-kB signaling pathway is broadly implicated in CLL cell survival and resistance to therapy, and proteasome inhibitors have been reported to block this pathway via inhibition of IkB degradation. We therefore investigated the impact of CFZ on NF-kB-mediated transcription in CLL patient cells. Paradoxically, our results indicate that CFZ leads to activation of NF-kB, as evidenced by increased nuclear accumulation of the p50 and p65 subunits of NF-kB, as well as phosphorylated IkBα. This correlates with enhanced binding of the p50/p65 heterodimer to an NF-kB probe in an electrophoretic mobility shift assay. However, despite this apparent NF-kB activation, no transcriptional increases were observed in NF-kB targets genes including Mcl-1, p53, Bcl-2, Bcl2A1 or XIAP. In addition, inhibition of NF-kB activity using a dominant negative (non-phosphorylatable) IkBα did not impair CFZ-induced cytotoxicity. This is the first study suggesting that treatment with a proteasome inhibitor induces a defective NF-κB response in CLL cells. The mechanism and relevance of this effect, as well as the pathway by which CFZ causes CLL cell death, continues to be investigated. Collectively, our data indicate that proteasome inhibition is a relevant therapeutic target in CLL and supports the development of carfilzomib for the treatment of this currently incurable disease.
Disclosures:
No relevant conflicts of interest to declare.
Involvement of CED-3/ICE Proteases in the Apoptosis of B-Chronic Lymphocytic Leukemia Cells
