B246 Sorafenib, also known as BAY 43-9006, is a multi-kinase inhibitor that induces apoptosis in human leukemia and other malignant cells. Recently, we demonstrated that sorafenib diminishes Mcl-1 protein expression by inhibiting translation through a MEK1/2/ERK1/2 signaling-independent mechanism, and that this phenomenon plays a key functional role in sorafenib lethality (Rahmani et al., Journal of Biologic Chemistry 280:35217-27, 2005). Here, we report that inducible expression of constitutively active MEK1 fails to protect U937 human leukemia cells from sorafenib-mediated lethality, indicating that sorafenib-induced cell death is unrelated to MEK1/2/ERK1/2 pathway inactivation. Notably, treatment with sorafenib induced ER stress manifested by immediate cytosolic calcium mobilization, GADD153 and GADD34 protein induction, PERK and eIF2α phosphorylation, XBP1 splicing, and a general reduction in protein synthesis as assessed by [ 35 S] methionine incorporation. Notably, genetic disruption of PERK (e.g., transfection of cells with DN-PERK), but not PKR, HRI, or CGN2, attenuated eIF2α phosphorylation, identifying PERK as the primary eIF2α kinase in this setting. These events were accompanied by the pronounced generation of reactive oxygen species (ROS) through a mechanism dependent upon cytosolic calcium mobilization and a significant decline in GRP78/Bip protein levels. Interestingly, enforced expression of IRE1α or XBP1 splice variant (XBP1s) significantly reduced sorafenib-mediated apoptosis, whereas knockdown of IRE1α or XBP1, disruption of PERK activity, or inhibition of eIF2α phosphorylation enhanced sorafenib-mediated lethality. Such observations suggest that these events comprise a cytoprotective component of the sorafenib-induced ER stress response. Finally, downregulation of caspase-2 or caspase-4 by siRNA or shRNA respectively significantly diminished apoptosis induced by sorafenib in these cells, providing further support for the notion that the lethal actions of sorafenib involve induction ER stress. Together, these findings demonstrate that ER stress represents a central component of a MEK1/2/ERK1/2-independent cell death program triggered by sorafenib. They also raise the possibility that induction of ER stress by sorafenib in human leukemia cells may play a role in determining interactions between this kinase inhibitor and other targeted agents.