laq824 and Leukemia

The role of reactive oxygen species (ROS) production on DNA damage and potentiation of fludarabine lethality by the histone deacetylase inhibitor (HDACI) LAQ-824 was investigated in human leukemia cells. Preexposure (24 h) of U937, HL-60, Jurkat, or K562 cells to LAQ-824 (40 nmol/L) followed by fludarabine (0.4 micromol/L) dramatically potentiated apoptosis (>or=75%). LAQ-824 triggered an early ROS peak (30 min-3 h), which declined by 6 h, following LAQ-824-induced manganese superoxide dismutase 2 (Mn-SOD2) upregulation. LAQ-824/fludarabine lethality was significantly diminished by either ROS scavengers N-acetylcysteine or manganese (III) tetrakis (4-benzoic acid) porphyrin or ectopic Mn-SOD2 expression and conversely increased by Mn-SOD2 antisense knockdown. During this interval, LAQ-824 induced early (4-8 h) increases in gamma-H2AX, which persisted (48 h) secondary to LAQ-824-mediated inhibition of DNA repair (e.g., down-regulation of Ku86 and Rad50, increased Ku70 acetylation, diminished Ku70 and Ku86 DNA-binding activity, and down-regulated DNA repair genes BRCA1, CHEK1, and RAD51). Addition of fludarabine further potentiated DNA damage, which was incompatible with cell survival, and triggered multiple proapoptotic signals including activation of nuclear caspase-2 and release of histone H1.2 into the cytoplasm. The latter event induced activation of Bak and culminated in pronounced mitochondrial injury and apoptosis. These findings provide a mechanistic basis for understanding the role of early HDACI-induced ROS generation and modulation of DNA repair processes in potentiation of nucleoside analogue-mediated DNA damage and lethality in leukemia. Moreover, they show for the first time the link between HDACI-mediated ROS generation and the recently reported DNA damage observed in cells exposed to these agents.

Topics: Acetylation; Antineoplastic Agents; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; Caspase 2; Cell Line, Tumor; Cytosol; DNA Damage; DNA Repair; Down-Regulation; Drug Screening Assays, Antitumor; Drug Synergism; Enzyme Activation; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Leukemia; Reactive Oxygen Species; Superoxide Dismutase; Vidarabine

Determinants of differentiation and apoptosis induction by the novel histone deacetylase inhibitor (HDACI) LAQ824 were examined in human leukemia cells (U937 and Jurkat). Exposure of U937 cells to a low concentration of LAQ824 (30 nM) resulted in a delayed (2 h) increase in reactive oxygen species (ROS), induction of p21(WAF1/CIP1), pRb dephosphorylation, growth arrest of cells in G(0)/G(1) phase, and differentiation. On the other hand, exposure of cells to a higher concentration of LAQ824 (75 nM) resulted in the early (30 min) generation of ROS, arrest of cells in G(2)/M phase, down-regulation of XIAP (at the transcriptional level) and Mcl-1 (through a caspase-mediated process), the acid sphingomyelinase-dependent generation of ceramide, and profound mitochondrial injury, caspase activation, and apoptosis. LAQ824-induced lethality in U937 cells did not involve the extrinsic apoptotic pathway, nor was it associated with death receptor up-regulation; instead, it was markedly inhibited by ectopic expression of Bcl-2, Bcl-x(L), XIAP, and Mcl-1. The free radical scavenger N-acetyl cysteine blocked LAQ824-mediated ROS generation, mitochondrial injury, Mcl-1 down-regulation, ceramide generation, and apoptosis, suggesting a primary role for oxidative injury in LAQ824 lethality. Together, these findings indicate that LAQ824-induced lethality represents a multifactorial process in which LAQ824-mediated ROS generation is necessary but not sufficient to induce apoptosis, and that the degree of XIAP and Mcl-1 down-regulation and ceramide generation determines whether this agent engages a maturation rather than an apoptotic program.

Topics: Apoptosis; Ceramides; Down-Regulation; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Jurkat Cells; Leukemia; Oxidative Stress; Reverse Transcriptase Polymerase Chain Reaction; Sphingomyelin Phosphodiesterase; U937 Cells; X-Linked Inhibitor of Apoptosis Protein

Human enhancer of zeste 2 (EZH2) protein belongs to the multiprotein polycomb repressive complex 2, which also includes suppressor of zeste 12 (SUZ12) and embryonic ectoderm development (EED). The polycomb repressive complex 2 complex possesses histone methyltransferase activity mediated by the Su(var)3-9, enhancer of zeste, and trithorax domain of EZH2, which methylates histone H3 on lysine (K)-27 (H3K27). In the present studies, we determined that treatment with the hydroxamate histone deacetylase inhibitor LBH589 or LAQ824 depleted the protein levels of EZH2, SUZ12, and EED in the cultured (K562, U937, and HL-60) and primary human acute leukemia cells. This was associated with decreased levels of trimethylated and dimethylated H3K27, with concomitant depletion of the homeobox domain containing HOXA9 and of MEIS1 transcription factors. Knockdown of EZH2 by EZH2 small interfering RNA also depleted SUZ12 and EED, inhibited histone methyltransferase activity, and reduced trimethylated and dimethylated H3K27 levels, with a concomitant loss of clonogenic survival of the cultured acute myelogenous leukemia (AML) cells. EZH2 small interfering RNA sensitized the AML cells to LBH589-mediated depletion of EZH2, SUZ12, and EED; loss of clonogenic survival; and LBH589-induced differentiation of the AML cells. These findings support the rationale to test anti-EZH2 treatment combined with hydroxamate histone deacetylase inhibitors as an antileukemia epigenetic therapy, especially against AML with coexpression of EZH2, HOXA9, and MEIS1 genes.

Topics: Acetylation; Acute Disease; Carrier Proteins; Chronic Disease; DNA-Binding Proteins; Down-Regulation; Enhancer of Zeste Homolog 2 Protein; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histone Deacetylases; Histone Methyltransferases; Histone-Lysine N-Methyltransferase; Histones; HL-60 Cells; Humans; Hydroxamic Acids; Indoles; K562 Cells; Leukemia; Neoplasm Proteins; Nuclear Proteins; Panobinostat; Polycomb Repressive Complex 2; Polycomb-Group Proteins; Protein Methyltransferases; Repressor Proteins; RNA, Small Interfering; Transcription Factors; U937 Cells

The hydroxamic acid (HAA) analogue pan-histone deacetylase (HDAC) inhibitors (HDIs) LAQ824 and LBH589 have been shown to induce acetylation and inhibit the ATP binding and chaperone function of heat shock protein (HSP) 90. This promotes the polyubiquitylation and degradation of the pro-growth and pro-survival client proteins Bcr-Abl, mutant FLT-3, c-Raf, and AKT in human leukemia cells. HDAC6 is a member of the class IIB HDACs. It is predominantly cytosolic, microtubule-associated alpha-tubulin deacetylase that is also known to promote aggresome inclusion of the misfolded polyubiquitylated proteins. Here we demonstrate that in the Bcr-abl oncogene expressing human leukemia K562 cells, HDAC6 can be co-immunoprecipitated with HSP90, and the knock-down of HDAC6 by its siRNA induced the acetylation of HSP90 and alpha-tubulin. Depletion of HDAC6 levels also inhibited the binding of HSP90 to ATP, reduced the chaperone association of HSP90 with its client proteins, e.g. Bcr-Abl, and induced polyubiquitylation and partial depletion of Bcr-Abl. Conversely, the ectopic overexpression of HDAC6 inhibited LAQ824-induced acetylation of HSP90 and alpha-tubulin and reduced LAQ824-mediated depletion of Bcr-Abl, AKT, and c-Raf. Collectively, these findings indicate that HDAC6 is also an HSP90 deacetylase. Targeted inhibition of HDAC6 leads to acetylation of HSP90 and disruption of its chaperone function, resulting in polyubiquitylation and depletion of pro-growth and pro-survival HSP90 client proteins including Bcr-Abl. Depletion of HDAC6 sensitized human leukemia cells to HAA-HDIs and proteasome inhibitors.

Topics: Acetylation; Antineoplastic Agents; Enzyme Inhibitors; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Histone Deacetylases; HSP90 Heat-Shock Proteins; Humans; Hydroxamic Acids; Indoles; K562 Cells; Leukemia; Molecular Chaperones; Neoplasm Proteins; Panobinostat; Protein Binding; Ubiquitin

Interactions between the novel histone deacetylase inhibitor LAQ824 and the cyclin-dependent kinase inhibitor roscovitine were examined in human leukemia cells. Pretreatment (24 hours) with a subtoxic concentration of LAQ824 (30 nmol/L) followed by a minimally toxic concentration of roscovitine (10 micromol/L; 24 hours) resulted in greater than additive effects on apoptosis in U937, Jurkat, and HL-60 human leukemia cells and blasts from three patients with acute myelogenous leukemia. These events were associated with enhanced conformational changes in Bax; mitochondrial release of cytochrome c, Smac/DIABLO, and apoptosis-inducing factor; and a marked increase in caspase activation. LAQ824/roscovitine-treated cells displayed caspase-dependent down-regulation of p21(CIP1) and Mcl-1 and a pronounced caspase-independent reduction in X-linked inhibitor of apoptosis (XIAP) expression. The lethality of this regimen was significantly attenuated by ectopic expression of XIAP, a nuclear localization signal-defective p21(CIP1) mutant, Mcl-1, and Bcl-2. Combined exposure to LAQ824 and roscovitine resulted in a significant reduction in XIAP mRNA levels and diminished phosphorylation of the carboxyl-terminal domain of RNA polymerase II. Notably, roscovitine blocked LAQ824-mediated differentiation. Finally, LAQ824 and roscovitine individually and in combination triggered an increase in generation of reactive oxygen species; moreover, coadministration of the free radical scavenger N-acetylcysteine prevented LAQ824/roscovitine-mediated mitochondrial injury and apoptosis. Collectively, these findings suggest that combined treatment of human leukemia cells with LAQ824 and roscovitine disrupts maturation and synergistically induces apoptosis, lending further support for an antileukemic strategy combining novel histone deacetylase and cyclin-dependent kinase inhibitors.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; Blotting, Western; Cell Cycle; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; Cytosol; Down-Regulation; Enzyme Inhibitors; Flow Cytometry; Histone Deacetylase Inhibitors; HL-60 Cells; Humans; Hydroxamic Acids; Jurkat Cells; Leukemia; Membrane Potentials; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Protein Conformation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Purines; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; Roscovitine; Time Factors; U937 Cells; X-Linked Inhibitor of Apoptosis Protein