Bcl-2 antagonists interact synergistically with bortezomib in DLBCL cells in association with JNK activation and induction of ER stress.

Mechanisms underlying interactions between the proteasome inhibitor bortezomib and small molecule Bcl-2 antagonists were examined in GC- and ABC-type human DLBCL (diffuse lymphocytic B-cell lymphoma) cells. Concomitant or sequential exposure to non- or minimally toxic concentrations of bortezomib or other proteasome inhibitors and either HA14-1 or gossypol resulted in a striking increase in Bax/Bak conformational change/translocation, cytochrome c release, caspase activation and synergistic induction of apoptosis in both GC- and ABC-type cells. These events were associated with a sharp increase in activation of the stress kinase JNK and evidence of ER stress induction (e.g., eIF2alpha phosphorylation, activation of caspases-2 and -4, and Grp78 upregulation). Pharmacologic or genetic (e.g., shRNA knockdown) interruption of JNK signaling attenuated HA14-1/bortezomib lethality and ER stress induction. Genetic disruption of the ER stress pathway (e.g., in cells expressing caspase-4 shRNA or DN-eIF2alpha) significantly attenuated lethality. The toxicity of this regimen was independent of ROS generation. Finally, HA14-1 significantly increased bortezomib-mediated JNK activation, ER stress induction, and lethality in bortezomib-resistant cells. Collectively these findings indicate that small molecule Bcl-2 antagonists promote bortezomib-mediated mitochondrial injury and lethality in DLBCL cells in association with enhanced JNK activation and ER stress induction. They also raise the possibility that such a strategy may be effective in different DLBCL sub-types (e.g., GC- or ABC), and in bortezomib-resistant disease.