BAY 43-9006 is a kinase inhibitor that induces apoptosis in a variety of tumor cells. Here we report that treatment with BAY 43-9006 results in marked cytochrome c and AIF release into the cytosol, caspase-9, -8, -7, and -3 activation, and apoptosis in human leukemia cells (U937, Jurkat, and K562). Pronounced apoptosis was also observed in blasts from patients with acute myeloid leukemia. The Ras/Raf/mitogen-activated protein kinase (MEK) 2 /extracellularsignal-regulated kinase (ERK) cascade plays a critical role in relaying signals from cell surface receptors to various cytoplasmic and nuclear proteins involved in diverse biological process such as cell growth, transformation, differentiation, and apoptosis (1). Aberrant activation of this pathway has been implicated in the development of many tumor types, and constitutive activation of this pathway has been observed in ϳ30% of all human cancer. The serine/threonine Raf kinase family, which consists of three proteins, C-Raf (also referred to as Raf-1), B-Raf, and A-Raf, is an essential component of this pathway (1, 2). Strikingly, B-Raf-activating mutations have been observed in ϳ70% of malignant melanomas (3, 4) and at lower frequencies in a number of other human cancer types, including colorectal (3, 5), ovarian, and papillary thyroid carcinomas (3, 6, 7). Moreover, overexpression of constitutively active c-Raf is sufficient to induce transformation of NIH 3T3 cells (8). Increased Raf/MEK/ERK activity has also been observed in a variety of leukemias, including acute myeloid leukemia (AML) and chronic myeloid leukemia (9, 10). In addition, constitutive activation of this pathway diminishes apoptosis in hematopoietic cells (11) and abrogates the cytokine dependence of several human and murine cytokine-dependent hematopoietic cells lines (e.g. TF-1, FDC-P1, and FL5.12) (12). Conversely, inhibition of this pathway by pharmacologic MEK inhibitors such as PD98059 or U0126 enhances apoptosis induction by a variety of agents, including paclitaxel (13) UCN01 (14), STI571 (15), proteasome inhibitors (16), and lovastatin (17). For these reasons, disrupting the Ras/Raf/MEK/ERK pathway represents an attractive anticancer strategy, particularly in leukemia cells.BAY 43-9006, a novel bi-aryl urea, has shown promising preclinical activity against a variety of tumor cell types and is currently undergoing phase II/III clinical evaluation (18 -20). Although it was initially developed as a specific inhibitor of C-Raf and B-Raf, subsequent studies revealed that this compound also inhibits several other important tyrosine kinases involved in tumor progression, including vascular epidermal growth factor receptor-2, vascular epidermal growth factor receptor-3, platelet-derived growth factor receptor-␤, Flt3, and c-Kit (21). Interestingly, BAY 43-9006 has been shown to inhibit C-Raf and wild type as well as mutant V600E B-Raf kinase activities in vitro and to diminish MEK/ERK activation in various tumor cell lines, including those harboring mutant Ras or B-Raf (21-23).Several studies...

Sorafenib is a multikinase 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-mediated lethality. Here, we report that inducible expression of constitutively active MEK1 fails to protect 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 endoplasmic reticulum (ER) stress in human leukemia cells (U937) manifested by immediate cytosolic-calcium mobilization, GADD153 and GADD34 protein induction, PKR-like ER kinase (PERK) and eukaryotic initiation factor 2␣ (eIF2␣) phosphorylation, XBP1 splicing, and a general reduction in protein synthesis as assessed by [35 S]methionine incorporation. These events were accompanied by pronounced generation of reactive oxygen species through a mechanism dependent upon cytosolic-calcium mobilization and a significant decline in GRP78/Bip protein levels. Interestingly, enforced expression of IRE1␣ markedly reduced sorafenib-mediated apoptosis, whereas knockdown of IRE1␣ or XBP1, disruption of PERK activity, or inhibition of eIF2␣ phosphorylation enhanced sorafenib-mediated lethality. Finally, downregulation of caspase-2 or caspase-4 by small interfering RNA significantly diminished apoptosis induced by sorafenib. Together, these findings demonstrate that ER stress represents a central component of a MEK1/2-ERK1/2-independent cell death program triggered by sorafenib.The discovery that aberrant activation of the Ras-Raf-MEK1/2-ERK1/2 module occurs in a high percentage (30%) of human cancers (12, 42, 48, 57) provided a rationale for attempting to disrupt this pathway as a candidate anticancer strategy. Recently, a number of specific inhibitors designed to interrupt this pathway at the level of Ras, Raf, or MEK1/2 have been developed. Among these, sorafenib, a biaryl urea, also known as BAY 43-9006 or Nexavar, was initially developed as a specific inhibitor of C-Raf and B-Raf. However, subsequent studies revealed that it also inhibits several other tyrosine kinases involved in tumor progression, including VEGFR-2, VEGFR-3, PDGFR-␤, Flt3, c-Kit, and Ret (6, 60). Interestingly, sorafenib has been shown to inhibit mutant (V600E) B-Raf kinase activities in vitro and to diminish MEK/extracellular signal-regulated kinase (ERK) activation in various tumor cell lines, including those harboring mutant Ras or B-Raf (25,56,60). Although this compound has potent activity in preclinical tumor xenograft models against a variety of tumor cell types (60), has shown promising activity in a number of clinical trials, and has recently been approved for the treatment of advanced renal cell carcinoma (51), the mechanism(s) by which it exerts its antitumor activity has not been fully elucidated and is currently the subject of ongoing i...