benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Leukemia--Myeloid--Acute

Activated CDC42-associated kinase-1 (ACK1/TNK2) and epigenetic regulators of the histone deacetylase (HDAC) family regulate the proliferation and survival of leukemic cells. 18 HDACs fall into four classes (I-IV). We tested the impact of clinically relevant histone deacetylase inhibitors (HDACi) on ACK1 and if such drugs combine favorably with the therapeutically used ACK1 inhibitor Dasatinib.. We applied the broad-range HDACi Panobinostat/LBH589 and the class I HDAC-specific inhibitor Entinostat/MS-275 to various acute and chronic myeloid leukemia cells (AML/CML). We also used the replicative stress inducer Hydroxyurea (HU), a standard drug for leukemic patients, and the apoptosis inducer Staurosporine (STS). To assess cytotoxic effects of HDACi, we measured cell cycle profiles and DNA fragmentation by flow cytometry. Western blot was employed to analyze protein expression and phosphorylation.. LBH589 and MS-275 induce proteolysis of ACK1 in CML and AML cells. Panobinostat more strongly induces apoptosis than Entinostat, and this correlates with a significantly pronounced loss of ACK1. STS and HU also propel the degradation of ACK1 in leukemic cells. Moreover, the caspase inhibitor z-VAD-FMK reduces ACK1 degradation in the presence of HDACi. Concomitant with the attenuation of ACK1, we noticed decreased phosphorylation of STAT3. Direct inhibition of ACK1 with Dasatinib also suppresses STAT3 phosphorylation. Furthermore, Dasatinib and HDACi combinations are effective against CML cells.. HDACs sustain the ACK1-STAT3 signaling node and leukemic cell growth. Consistent with their different effects on ACK1 stability or auto-phosphorylation, Dasatinib and HDACi combinations produce beneficial antileukemic effects.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Caspases; Dasatinib; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Indoles; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Panobinostat; Phosphorylation; Protein-Tyrosine Kinases; STAT3 Transcription Factor

Inhibitor of Apoptosis (IAP) proteins are expressed at high levels in acute myeloid leukemia (AML) and contribute to resistance to programmed cell death. Here, we report that inhibition of IAP proteins by the small-molecule Smac mimetic BV6 acts together with histone deacetylase (HDAC) inhibitors (HDACIs) such as MS275 or SAHA to trigger cell death in AML cell lines in a synergistic manner, as underscored by calculation of combination index (CI). Also, BV6 and HDACIs cooperate to trigger DNA fragmentation, a marker of apoptotic cell death, and to suppress long-term clonogenic survival of AML cells. In contrast, equimolar concentrations of BV6 and MS275 or SAHA do not synergize to elicit cell death in normal peripheral blood lymphocytes (PBLs), emphasizing some tumor cell selectivity of this combination treatment. Addition of the tumor necrosis factor (TNF)α-blocking antibody Enbrel significantly reduces BV6/MS275-induced cell death in the majority of AML cell lines, indicating that autocrine/paracrine TNFα signaling contributes to cell death. Remarkably, the broad-range caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) fails to rescue MV4-11, Molm13 and OCI-AML3 cells and even enhances BV6/MS275-mediated cell death, whereas zVAD.fmk reduces BV6/MS275-induced cell death in NB4 cells. Annexin-V/propidium iodide (PI) double staining reveals that BV6/MS275 cotreatment predominately increases the percentage of double-positive cells. Of note, the Receptor-Interacting Protein (RIP)1 inhibitor necrostatin-1 (Nec-1) or the Mixed Lineage Kinase Domain-Like protein (MLKL) inhibitor necrosulfonamide (NSA) significantly reduce BV6/MS275-induced cell death in the presence of zVAD.fmk, suggesting that BV6/MS275 cotreatment triggers necroptosis when caspases are inhibited. Thus, BV6 acts in concert with HDACIs to induce cell death in AML cells and can bypass apoptosis resistance, at least in several AML cell lines, by engaging necroptosis as an alternative route of regulated cell death. The identification of a novel synergism of BV6 and HDACIs has important implications for the development of new treatment strategies for AML.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspases; Cell Line, Tumor; Drug Synergism; Histone Deacetylase Inhibitors; Humans; Leukemia, Myeloid, Acute; Oligopeptides; Signal Transduction; Tumor Necrosis Factor-alpha

In t(8;21) acute myeloid leukemia (AML), the AML1/ETO fusion protein promotes leukemogenesis by recruiting class I histone deacetylase (HDAC)-containing repressor complex to the promoter of AML1 target genes. Valproic acid (VPA), a commonly used antiseizure and mood stabilizer drug, has been shown to cause growth arrest and induce differentiation of malignant cells via HDAC inhibition. VPA causes selective proteasomal degradation of HDAC2 but not other class I HDACs (i.e., HDAC 1, 3, and 8). Therefore, we raised the question of whether this drug can effectively target the leukemogenic activity of the AML1/ETO fusion protein that also recruits HDAC1, a key regulator of normal and aberrant histone acetylation. We report here that VPA treatment disrupts the AML1/ETO-HDAC1 physical interaction, stimulates the global dissociation of AML1/ETO-HDAC1 complex from the promoter of AML1/ETO target genes, and induces relocation of both AML1/ETO and HDAC1 protein from nuclear to perinuclear region. Furthermore, we show that mechanistically these effects associate with a significant inhibition of HDAC activity, histone H3 and H4 hyperacetylation, and recruitment of RNA polymerase II, leading to transcriptional reactivation of target genes (i.e., IL-3) otherwise silenced by AML1/ETO fusion protein. Ultimately, these pharmacological effects resulted in significant antileukemic activity mediated by partial cell differentiation and caspase-dependent apoptosis. Taken together, these data support the notion that VPA might effectively target AML1/ETO-driven leukemogenesis through disruption of aberrant HDAC1 function and that VPA should be integrated in novel therapeutic approaches for AML1/ETO-positive AML.

Topics: Acetylation; Amino Acid Chloromethyl Ketones; Apoptosis; Caspase 3; Caspase 9; Cell Differentiation; Cell Line, Tumor; Cell Nucleus; Chromatin Assembly and Disassembly; Chromatin Immunoprecipitation; Core Binding Factor Alpha 2 Subunit; Cysteine Proteinase Inhibitors; DNA; Gene Expression Regulation, Leukemic; Histone Deacetylase 1; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Interleukin-3; Leukemia, Myeloid, Acute; Oncogene Proteins, Fusion; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Promoter Regions, Genetic; Protein Binding; Repressor Proteins; RNA Polymerase II; RUNX1 Translocation Partner 1 Protein; Valproic Acid

To explore the possible cross-talk between the IL-6 and TGF-beta1 pathways in AML blast cells, the effect of TGF-beta1 pretreatment on IL-6-induced STAT3 tyrosine phosphorylation was studied. A reduction of STAT3 tyrosine phosphorylation after TGF-beta1 pretreatment was observed in four out of 40 AML cases (10%), although all of the AML cases responded to TGF-beta1 by means of SMAD3 translocation. The reduced IL-6-mediated STAT3 tyrosine phosphorylation after pre-treatment with TGF-beta1 was associated with apoptosis and coincided with the degradation of certain cellular proteins, including JAK1 and -2 and Tyk2, without affecting the ERK expression and phosphorylation. Furthermore, treatment of AML blasts with the cytostatic agent VP16, as an alternative way to induce apoptosis, resulted in a similar degree of degradation of JAK kinases and concomitant reduction of IL-6-mediated STAT3 tyrosine phosphorylation. Although degradation of JAK kinases could be rescued by incubating the cells with the pan-caspase inhibitor Z-VAD-fmk, the attenuating effect of TGF-beta1 treatment on the STAT3 tyrosine phosphorylation was still partly present. It was shown that in AML cells cultured in the presence of Z-VAD-fmk, TGF-beta1 pretreatment resulted in a reduction of JAK1 phosphorylation upon IL-6 stimulation. Expression of SOCS1 and -3 could be ruled out as a possible cause of reduced JAK1 phosphorylation levels in the investigated AML case.

Topics: Amino Acid Chloromethyl Ketones; Annexin A5; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Carrier Proteins; Caspase 3; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Down-Regulation; Electrophoretic Mobility Shift Assay; Epithelial Cells; Etoposide; Humans; Interleukin-6; Intracellular Signaling Peptides and Proteins; Janus Kinase 1; Leukemia, Myeloid, Acute; Phosphorylation; Protein Transport; Protein-Tyrosine Kinases; Repressor Proteins; Signal Transduction; Smad3 Protein; STAT3 Transcription Factor; Suppressor of Cytokine Signaling 1 Protein; Suppressor of Cytokine Signaling Proteins; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured; Tyrosine

The ELL gene encodes an RNA polymerase II transcription factor that frequently undergoes translocation with the MLL gene in acute human myeloid leukemia. Here, we report that ELL can regulate cell proliferation and survival. In order to better understand the physiological role of the ELL protein, we have developed an ELL-inducible cell line. Cells expressing ELL were uniformly inhibited for growth by a loss of the G(1) population and an increase in the G(2)/M population. This decrease in cell growth is followed by the condensation of chromosomal DNA, activation of caspase 3, poly(ADP ribose) polymerase cleavage, and an increase in sub-G(1) population, which are all indicators of the process of programmed cell death. In support of the role of ELL in induction of cell death, expression of an ELL antisense RNA or addition of the caspase inhibitor ZVAD-fmk results in a reversal of ELL-mediated death. We have also demonstrated that the C-terminal domain of ELL, which is conserved among the ELL family of proteins that we have cloned (ELL, ELL2, and ELL3), is required for ELL's activity in the regulation of cell growth. These novel results indicate that ELL can regulate cell growth and survival and may explain how ELL translocations result in the development of human malignancies.

Topics: Amino Acid Chloromethyl Ketones; Antigens, Differentiation; Apoptosis; Blotting, Western; Caspase 3; Caspase Inhibitors; Caspases; Cell Cycle Proteins; Cell Death; Cell Division; Cell Line; Cell Survival; Cysteine Proteinase Inhibitors; DNA; DNA-Binding Proteins; Enzyme Activation; Enzyme Inhibitors; Flow Cytometry; G1 Phase; G2 Phase; Humans; Leukemia, Myeloid, Acute; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Mitosis; Neoplasm Proteins; Oligonucleotides, Antisense; Peptide Elongation Factors; Plasmids; Poly(ADP-ribose) Polymerases; Propidium; Protein Phosphatase 1; Proteins; RNA, Messenger; Time Factors; Transcription Factors; Transcriptional Elongation Factors; Transfection; Translocation, Genetic; Tumor Cells, Cultured