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)

Effects of the PI-3 kinase inhibitor LY294002 (LY) have been examined in relation to responses of human leukemia cells to histone deacetylase inhibitors (HDIs). Coexposure of U937 cells for 24 h to marginally toxic concentrations of LY294002 (e.g., 30 lm) and sodium butyrate (SB; 1 mm) resulted in a marked increase in mitochondrial damage (e.g., cytochrome c and Smac/DIABLO release, loss of DW m ), caspase activation, and apoptosis. Similar results were observed in Jurkat, HL-60, and K562 leukemic cells and with other HDIs (e.g., SAHA, MS-275). Exposure of cells to SB/LY was associated with Bcl-2 and Bid cleavage, XIAP and Mcl-1 downregulation, and diminished CD11b expression. While LY blocked SBmediated Akt activation, enforced expression of a constitutively active (myristolated) Akt failed to attenuate SB/LY-mediated lethality. Unexpectedly, treatment of cells with SB7LY resulted in a marked reduction in phosphorylation (activation) of p44/42 mitogen-activated protein (MAP) kinase. Moreover, enforced expression of a constitutively active MEK1 construct partially but significantly attenuated SB/LY-induced apoptosis. Lastly, cotreatment with LY blocked SB-mediated induction of p21 CIP1/WAF1 ; moreover, enforced expression of p21 CIP1/WAF1 significantly reduced SB/LY-mediated apoptosis. Together, these findings indicate that LY promotes SBmediated apoptosis through an AKT-independent process that involves MEK/MAP kinase inactivation and interference with p21 CIP1/WAF1 induction.

Signal transduction events regulating induction of apoptosis by the histone deacetylase inhibitors (HDIs) sodium butyrate (SB) and SAHA have been examined in Bcr/Abl + human leukemia cells (K562, LAMA 84). Exposure of K562 cells to ≥ 3.0 mM SB or 3.0 µM SAHA for 24-48 hr resulted in a marked induction of mitchondrial damage (e.g., cytochrome c release) and apoptosis, events associated with downregulation of Bcr/Abl and Raf-1, induction of p21 CIP1 , inactivation of MEK1/2, ERK1/2, and p70 S6K , and a dramatic increase in JNK activation. HDI-mediated apoptosis was attenuated by pharmacologic JNK inhibitors and enhanced by the MEK1/2 inhibitor U0126 as well as by the JNK activator anisomycin. Interestingly, HDI-induced JNK activation was potentiated by pharmacologic MEK inhibition. Furthermore, HDI lethality was significantly diminished in cells ectopically expressing constitutively active MEK1, confirming a functional role for MEK/ERK inactivation in HDI-mediated apoptosis. Similar events were observed in Bcr/Abl + LAMA 84 cells. Lastly, the free radical scavenger L-N-acetylcysteine (LNAC) attenuated HDI-mediated ROS generation, JNK activation, and apoptosis. Together, these findings support a model in which induction of apoptosis in Bcr/Abl + cells by HDIs involves coordinate inactivation of the cytoprotective Raf/MEK/ERK pathway in conjunction with the ROS-dependent activation of JNK.

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.