Potentiation of Bortezomib-Induced Killing of Lymphoma Cells by Inhibition of Autophagy and Prevention of I-κBα Degradation

Abstract

AbstractAbstract 116Previous studies have shown that germinal center B cell-like (GCB) diffuse large B-cell lymphoma (DLBCL) are resistant to proteasome inhibitors such as bortezomib. The mechanism by which GCB-DLBCL cells escape from proteasome inhibitor-induced apoptosis is unclear. To investigate this further, we examined the proteasomal pathway, expression of anti-apoptotic proteins and autophagy. Using bortezomib or MG-262, we show that DLBCL cells have differential sensitivity to proteasome inhibitor-mediated cell death, even though the effects on proteasome inhibition were similar. DLBCL cells that either over-express anti-apoptotic proteins such as Bcl-2, or lack pro-apoptotic proteins including Bax/Bak, are more resistant to proteasome inhibitor-induced reduction of mitochondrial membrane potential and activation of caspase-3. Treatment with bortezomib induced autophagy in both sensitive and resistant DLBCL cells, as demonstrated by an accumulation and aggregation of the autophagy marker protein LC3-II. Bortezomib induced accumulation of ubiquitinated proteins and a decrease in the adaptor protein p62, indicating activation of autophagic degradation. Fluorescent microscopy revealed that bortezomib induced p62 recruits both ubiquitinated proteins and LC3-II, suggesting that p62 leads ubiquitinated protein to autophagosomes. Treatment with bortezomib thereby promotes I-kBα degradation, demonstrating that the route of I-κBα degradation is not via the ubiquitin-proteasome degradation system. Bortezomib-induced I-kBα degradation was detected in both DLBCL cell lines and primary DLBCL and follicular lymphoma samples. Chloroquine (CQ), an inhibitor of autophagy, significantly increased bortezomib-induced accumulation of p62 and ubiquitinated proteins, including I-κBα, Bax and p53. CQ alone induced a dose-dependent increase in I-kBα protein levels, indicating that I-κBα protein can be degraded by autophagy even in the absence of proteasome inhibition. Importantly, the combination of proteasome and autophagy inhibitors shows great potential to kill apoptosis-resistant lymphoma cells. Proteasome inhibitor with or without CQ induced cell death in DLBCL cells cannot be blocked completely by either caspase inhibitors or knockdown of Bax/Bak proteins, suggesting that cell death occurs via a caspase-independent pathway. In summary, proteasome inhibitors induce autophagy and confer DLBCL cells resistance by eliminating I-κBα and possibly other pro-apoptotic proteins. Addition of autophagy inhibitors to bortezomib has the potential to induce increased killing in patients with resistant lymphoma. Disclosures:Gribben:Roche: Consultancy; Celgene: Consultancy; GSK: Honoraria; Napp: Honoraria.