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...

Interactions between histone deacetylase inhibitors (HDACIs) and the alkyl-lysophospholipid perifosine were examined in human leukemia cells. Coadministration of sodium butyrate, suberoylanilide hydroxamic acid (SAHA), or trichostatin with perifosine synergistically induced mitochondrial dysfunction (cytochrome c and apoptosis-inducing factor release), caspase-3 and -8 activation, apoptosis, and a marked decrease in cell growth in U937 as well as HL-60 and Jurkat leukemia cells. These events were associated with inactivation of extracellular signal-regulated kinase (ERK) 1/ 2 and Akt, p46 c-jun-NH 2 -kinase (JNK) activation, and a pronounced increase in generation of ceramide and reactive oxygen species (ROS). They were also associated with upregulation of Bak and a marked conformational change in Bax accompanied by membrane translocation. Ectopic expression of Bcl-2 delayed but was ultimately ineffective in preventing perifosine/HDACI-mediated apoptosis. Enforced expression of constitutively active mitogen-activated protein kinase kinase (MEK) 1 or myristoylated Akt blocked HDACI/perifosine-mediated ceramide production and cell death, suggesting that MEK/ERK and Akt inactivation play a primary role in these phenomena. However, inhibition of JNK activation (e.g., by the JNK inhibitor SP600125) did not attenuate sodium butyrate/perifosineinduced apoptosis. In addition, the free radical scavenger Nacetyl-L-cysteine attenuated ROS generation and apoptosis mediated by combined treatment. Finally, the acidic sphingomyelinase inhibitor desipramine attenuated HDACI/ perifosine-mediated ceramide and ROS production as well as cell death. Together, these findings indicate that coadministration of HDACIs with perifosine in human leukemia cells leads to Akt and MEK/ERK disruption, a marked increase in ceramide and ROS production, and a striking increase in mitochondrial injury and apoptosis. They also raise the possibility that combining these agents may represent a novel antileukemic strategy. (Cancer Res 2005; 65(6): 2422-32)

Interactions between the histone deacetylase (HDAC) inhibitors suberanoylanilide hydroxamic acid (SAHA) and sodium butyrate (SB) and the heat shock protein (Hsp) 90 antagonist 17-allylamino 17-demethoxygeldanamycin (17-AAG) have been examined in Bcr-Abl ϩ human leukemia cells (K562 and LAMA84), including those sensitive and resistant to STI571 (imatinib mesylate). Cotreatment with 17-AAG and SAHA or SB synergistically induced mitochondrial dysfunction (cytochrome c and apoptosis-inducing factor release), caspase-3 and -8 activation, apoptosis, and growth inhibition. Similar effects were observed in LAMA84 cells and K562 cells resistant to STI571, as well as in CD34 ϩ cells isolated from the bone marrows of three patients with chronic myelogenous leukemia. These events were associated with increased binding of BcrAbl, Raf-1, and Akt to Hsp70, and inactivation of extracellular signal-regulated kinase 1/2 and Akt. In addition, 17-AAG/SAHA abrogated the DNA binding and the transcriptional activities of signal transducer and activator of transcription (STAT) 5 in K562 cells, including those ectopically expressing a constitutively active STAT5A construct. Cotreatment with 17-AAG and SAHA also induced down-regulation of Mcl-1, Bcl-xL, and BRaf; up-regulation of Bak; cleavage of 14-3-3 proteins; and a profound conformational change in Bax accompanied by translocation to the membrane fraction. Moreover, ectopic expression of Bcl-2 attenuated cell death induced by this regimen, implicating mitochondrial injury in the lethality observed. Together, these findings raise the possibility that combining HDAC inhibitors with the Hsp90 antagonist 17-AAG may represent a novel strategy against Bcr-Abl ϩ leukemias, including those resistant to STI571.17-Allylamino 17-demethoxygeldanamycin (17-AAG), a derivative of the ansamycin antibiotic geldanamycin, has shown promising preclinical activity against a variety of tumor cell types and is currently undergoing phase II clinical evaluation (Dunn, 2002). 17-AAG antineoplastic activity has been attributed to binding to the ATP/ADP binding pocket of the heat shock protein (Hsp) 90, thereby inhibiting its function as a molecular chaperone. Hsp90 plays a key role in the intracellular trafficking and maturation of diverse cell signaling proteins, including those involved in cell survival such as Bcr-Abl, Raf-1, ErbB2, and Akt.

Interactions between the histone deacetylase inhibitors (HDACIs) suberanoylanilide hydroxamic acid (SAHA) and sodium butyrate (SB) and the heat shock protein 90 (Hsp90) antagonist 17-AAG have been examined in Bcr-Abl+ human leukemia cells (K562 and LAMA84), including those sensitive and resistant to STI571. Co-administration of 17-AAG with SAHA or SB for 24 h synergistically induced mitochondrial dysfunction (cytochrome c and AIF release), caspase-3 and -8 activation, apoptosis, and growth inhibition. Similar effects were observed in LAMA84 cells and K562 cells resistant to STI-571 as well as in CD34+ leukemic cells obtained from patients with CML including those who had developed a resistance against STI-571. These events were associated with increased association of Bcr/abl, Raf-1, and Akt with Hsp70, whereas the total level of these proteins markedly decreased. A pronounced inactivation of ERK1/2, and activation of JNK were also observed. In addition, 17-AAG/SAHA abrogated DNA binding and transcriptional activities of STAT5 in K562 cells, including those ectopically expressing a constitutively active STAT5A construct. Co-administration of 17-AAG and SAHA also induced downregulation of Mcl-1, Bcl-xL, B-Raf, and p21CIP1, upregulation of Bak, cleavage of 14-3-3 proteins, and a profound conformational change in Bax accompanied by translocation to the membrane fraction. Together, these findings raise the possibility that combining HDACIs with the Hsp90 antagonist 17-AAG may represent a novel strategy against Bcr-Abl+ leukemias, including those resistant to STI571.