The PRC2 complex protein EZH2 is a histone methyltransferase that is known to bind and recruit DNMT1 to the DNA to modulate DNA methylation. Here, we determined that the pan-HDAC inhibitor ...panobinostat (LBH589) treatment depletes DNMT1 and EZH2 protein levels, disrupts the interaction of DNMT1 with EZH2, as well as de-represses JunB in human acute leukemia cells. Similar to treatment with the hsp90 inhibitor 17-DMAG, treatment with panobinostat also inhibited the chaperone association of heat shock protein 90 with DNMT1 and EZH2, which promoted the proteasomal degradation of DNMT1 and EZH2. Unlike treatment with the DNA methyltransferase inhibitor decitabine, which demethylates JunB promoter DNA, panobinostat treatment mediated chromatin alterations in the JunB promoter. Combined treatment with panobinostat and decitabine caused greater attenuation of DNMT1 and EZH2 levels than either agent alone, which was accompanied by more JunB de-repression and loss of clonogenic survival of K562 cells. Co-treatment with panobinostat and decitabine also caused more loss of viability of primary AML but not normal CD34+ bone marrow progenitor cells. Collectively, these findings indicate that co-treatment with panobinostat and decitabine targets multiple epigenetic mechanisms to de-repress JunB and exerts antileukemia activity against human acute myeloid leukemia cells.
Inhibitor of Growth (ING) proteins are epigenetic "readers" that recognize trimethylated lysine 4 of histone H3 (H3K4Me3) and target histone acetyl transferase (HAT) and histone deacetylase (HDAC) ...complexes to chromatin.
Here we asked whether dysregulating two epigenetic pathways with chemical inhibitors showed synergistic effects on breast cancer cell line killing. We also tested whether ING1 could synergize better with chemotherapeutics that target the same epigenetic mechanism such as the HDAC inhibitor LBH589 (Panobinostat) or a different epigenetic mechanism such as 5-azacytidine (5azaC), which inhibits DNA methyl transferases. Simultaneous treatment of breast cancer cell lines with LBH589 and 5azaC did not show significant synergy in killing cells. However, combination treatment of ING1 with either LBH589 or 5azaC did show synergy. The combination of ING1b with 5azaC, which targets two distinct epigenetic mechanisms, was more effective at lower doses and enhanced apoptosis as determined by Annexin V staining and cleavage of caspase 3 and poly-ADP-ribose polymerase (PARP). ING1b plus 5azaC also acted synergistically to increase γH2AX staining indicating significant levels of DNA damage were induced. Adenoviral delivery of ING1b with 5azaC also inhibited cancer cell growth in a murine xenograft model and led to tumor regression when viral concentration was optimized in vivo.
These data show that targeting distinct epigenetic pathways can be more effective in blocking cancer cell line growth than targeting the same pathway with multiple agents, and that using viral delivery of epigenetic regulators can be more effective in synergizing with a chemical agent than using two chemotherapeutic agents. This study also indicates that the ING1 epigenetic regulator may have additional activities in the cell when expressed at high levels.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is a multidomain protein that plays a critical role in maintaining DNA methylation patterns through concurrent recognition of hemimethylated ...DNA and histone marks by various domains, and recruitment of DNA methyltransferase 1 (DNMT1). UHRF1 is overexpressed in various cancers, including breast cancer. The tandem tudor domain (TTD) of UHRF1 specifically and tightly binds to histone H3 di- or trimethylated at lysine 9 (H3K9me2 or H3K9me3, respectively), and this binding is essential for UHRF1 function. We developed an H3K9me3 peptide displacement assay, which was used to screen a library of 44,000 compounds for small molecules that disrupt the UHRF1-H3K9me3 interaction. This screen resulted in the identification of NV01, which bound to UHRF1-TTD with a K
value of 5 μM. The structure of UHRF1-TTD in complex with NV01 confirmed binding to the H3K9me3-binding pocket. Limited structure-based optimization of NV01 led to the discovery of NV03 (K
of 2.4 μM). These well-characterized small-molecule antagonists of the UHRF1-H3K9me2/3 interaction could be valuable starting chemical matter for developing more potent and cell-active probes toward further characterizing UHRF1 function, with possible applications as anticancer therapeutics.
Abstract
The Polycomb Repressive Complex-2 (PRC2) plays important roles in regulating gene expression through its histone H3 lysine methyl transferase activity. Dysregulation of PRC2 is observed in ...multiple cancers. For example, gain of function mutations of the EZH2 catalytic subunit of PRC2 have been reported in B-cell lymphomas, melanoma and parathyroid carcinoma. Components of the complex are also overexpressed in a number of cancers. The PRC2-complex have therefore become a major target for anti-cancer drug discovery. We and others, have reported anti-tumor activity of small molecular weight agents targeting the SAM-binding pocket of EZH2 in preclinical models and a number of EZH2 inhibitors ore showing promising activity in clinical trials. EED and SUZ12 are two additional components of the PRC2 core complex required for regulation of its catalytic function. High resolution structures and other reports have showed that binding of EED to tri-methylated histone H3 Lysine 27 (H3K27Me3) further stimulates the catalytic effect of EZh2. This suggests that small molecular weight compounds binding to and interfering with the EED stimulatory effect on Ezh2 may be able to modulate PRC2 activity. Following this possibility, we launched a comprehensive effort to discover non-catalytic site PRC2 binders. The discovery and characterization of EED226, a highly potent and selective EED-binding inhibitor of PRC2 will be presented. Data will be presented to demonstrate that EED226 inhibits PRC2 function via a distinct allosteric mechanism by binding to the H3K27Me3 binding pocket of EED. Furthermore, EED226 potently inhibits cellular H3K27Me3 levels, modulates target gene expression similar to the SAM competitive Ezh2 inhibitors and inhibits the proliferation of cell lines carrying mutant Ezh2. Oral administration of EED226 to EZH2-mutant tumor bearing mice leads to profound tumor regression. Our data therefore demonstrates that the multi-subunit PRC2 complex can be targeted at non-catalytic binding pockets to achieve anti-tumor activity. Some unique features of the anti-tumor activity achieved through targeting PRC2 will be discussed.
Citation Format: Peter W. Atadja. Targeting the PRC2 complex through EED for anti-cancer therapy abstract. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr IA19.
Purpose: Mutant FLT-3 receptor tyrosine kinase is a client protein of the molecular chaperone heat shock protein 90 and is commonly
present and contributes to the leukemia phenotype in acute ...myelogenous leukemia (AML). LAQ824, a cinnamyl hydroxamate histone
deacetylase inhibitor, is known to induce acetylation and inhibition of heat shock protein 90. Here, we determined the effects
of LAQ824 and/or PKC412 (a FLT-3 kinase inhibitor) on the levels of mutant FLT-3 and its downstream signaling, as well as
growth arrest and cell-death of cultured and primary human AML cells.
Experimental Design: The effect of LAQ824 and/or PKC412 treatment was determined on the levels of FLT-3 and phosphorylated (p)-FLT-3, on downstream
pro-growth and pro-survival effectors, e.g. , p-STAT5, p-AKT, and p-extracellular signal-regulated kinase (ERK) 1/2, and on the cell cycle status and apoptosis in the
cultured MV4–11 and primary AML cells with mutant FLT-3.
Results: Treatment with LAQ824 promoted proteasomal degradation and attenuation of the levels of FLT-3 and p-FLT-3, associated with
cell cycle G 1 -phase accumulation and apoptosis of MV4–11 cells. This was accompanied by attenuation of p-STAT5, p-AKT, and p-ERK1/2 levels.
STAT-5 DNA-binding activity and the levels of c-Myc and oncostatin M were also down-regulated. Cotreatment with LAQ824 and
PKC412 synergistically induced apoptosis of MV4–11 cells and induced more apoptosis of the primary AML cells expressing mutant
FLT-3. This was also associated with more attenuation of p-FLT-3, p-AKT, p-ERK1/2, and p-STAT5.
Conclusions: The combination of LAQ824 and PKC412 is highly active against human AML cells with mutant FLT-3, which merits in vivo studies of the combination against human AML.
Purpose: Histone deacetylase inhibitors have emerged as potent anticancer compounds. Using a nude-mouse xenograft model, for the first
time we evaluated the response of human gastrointestinal stromal ...tumors (GIST) carrying different oncogenic KIT mutations to panobinostat (LBH589), administered single or in combination with imatinib.
Experimental Design: We grafted the human GIST882 cell line with KIT exon 13 mutation and two biopsies from patients radiologically progressing under imatinib showing KIT exon11 and KIT exon9 mutations, respectively. Our study included 4 groups: A ( n = 9; control), B ( n = 10; panobinostat 10 mg/kg daily, i.p.), C ( n = 9; imatinib 150 mg/kg bidaily, p.o), and D ( n = 8; combination panobinostat-imatinib, same dose/schedule as above). Treatment lasted 12 days. Tumor size was measured regularly
using standard variables. Histopathological assessment was by H&E, and immunohistochemically with KIT, cleaved caspase-3,
Ki-67, and histone acetylation staining.
Results: Overall, GIST xenografts responded rapidly to panobinostat as indicated by tumor regression, necrosis, hemorrhages, fibrosis,
and/or myxoid degeneration, remarkable apoptosis, and substantial decline of cell proliferation. H3 and H4 acetylation increased
significantly from control level in all treated groups. The combination of panobinostat and imatinib further enhanced most
of the assessed parameters.
Conclusions: We show for the first time potential therapeutic activity of panobinostat in human GISTs, in vivo . Our results warrant further exploration of histone deacetylase inhibitors for the treatment of advanced GISTs. Our study
is also the first one on human GIST mouse xenografts established using patient biopsies.
Generation of hepatocyte from embryonic stem cells (ESCs) holds great promise for hepatocyte replacement therapy to treat liver diseases. Achieving high efficiency of directed differentiation of ESCs ...to hepatocyte is of critical importance. Previously, Wnt3a has been reported to promote Activin A-induced human definitive endoderm (DE) differentiation, the early stage of hepatocyte differentiation. However, the underlying molecular mechanisms are not clear. Growing evidence demonstrated that microRNAs (miRNAs) are key regulators involved in various important biological processes including the regulation of stem cell differentiation. In the present study, we profiled genome wide miRNA expression during Wnt3a and Activin A induced mouse DE differentiation. We uncovered distinct miRNA expression patterns during DE differentiation with the identification of a subset of miRNAs whose expression is synergistically regulated by Wnt3a/Activin A treatment at different stages of DE differentiation. Forced expression of a pool of such synergistically regulated miRNAs alone could partially promote DE differentiation, indicating a regulatory role of them. Using TargetScan and GeneGO pathway analyses, the synergistically regulated miRNAs are predicted to regulate key pathways involved in DE differentiation; among them includes the regulation of histone acetylation. Consistently, Wnt3a and Activin A treatment increased global histone acetylation which can be partially mimicked by over expression of the pooled miRNAs. Chromatin IP (ChIP) experiments demonstrated that the promoter regions of Sox17 and Foxa2 are subjected to histone acetylation regulation. Administration of Hdac inhibitors greatly augmented DE differentiation. Our data uncovered a novel epigenetic mechanism of Wnt3a and Activin A induced DE differentiation, whereby the treatment of growth factors induced histone acetylation at least in part by the regulation of miRNA expression.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Androgen receptor plays a critical role in the development of primary as well as advanced hormone-refractory prostate cancer.
Therefore, ablation of androgen receptor from prostate cancer cells is an ...interesting concept for developing a new therapy
not only for androgen-dependent prostate cancer but also for metastatic hormone-refractory prostate cancer, for which there
is no effective treatment available. We report here that LAQ824, a cinnamyl hydroxamatic acid histone deacetylase inhibitor
currently in human clinical trials, effectively depleted androgen receptor in prostate cancer cells at nanomolar concentrations.
LAQ824 seemed capable of depleting both the mutant and wild-type androgen receptors in either androgen-dependent and androgen-independent
prostate cancer cells. Although LAQ824 may exert its effect through multiple mechanisms, several lines of evidence suggest
that inactivation of the heat shock protein-90 (Hsp90) molecular chaperone is involved in LAQ824-induced androgen receptor
depletion. Besides androgen receptor, LAQ824 reduced the level of Hsp90 client proteins HER-2 (ErbB2), Akt/PKB, and Raf-1
in LNCaP cells. Another Hsp90 inhibitor, 17-allyamino-17-demethoxygeldanamycin (17-AAG), also induced androgen receptor diminution.
LAQ824 induced Hsp90 acetylation in LNCaP cells, which resulted in inhibition of its ATP-binding activity, dissociation of
Hsp90-androgen receptor complex, and proteasome-mediated degradation of androgen receptor. Consequently, LAQ824 blocked androgen-induced
prostate-specific antigen production in LNCaP cells. LAQ824 effectively inhibited cell proliferation and induced apoptosis
of these prostate cancer cells. These results reveal that LAQ824 is a potent agent for depletion of androgen receptor and
a potential new drug for prostate cancer.
Purpose: To evaluate the effects of combining the multiple receptor tyrosine kinase inhibitor AEE788 and histone deacetylase (HDAC)
inhibitors on cytotoxicity in a broad spectrum of cancer cell ...lines, including cisplatin-resistant ovarian adenocarcinoma
cells.
Experimental Design: Multiple cancer cell lines were treated in vitro using AEE788 and HDAC inhibitors (LBH589, LAQ824, and trichostatin A), either alone or in combination. Effects on cytotoxicity
were determined by growth and morphologic assays. Effects of the combination on cell signaling pathways were determined by
Western blotting, and the results were confirmed using pathway-specific inhibitors and transfection of constitutively active
proteins.
Results: Cell treatment with AEE788 and HDAC inhibitors (LBH589, LAQ824, and trichostatin A) in combination resulted in synergistic
induction of apoptosis in non–small cell lung cancer (MV522, A549), ovarian cancer (SKOV-3), and leukemia (K562, Jurkat, and
ML-1) cells and in OV202hp cisplatin-resistant human ovarian cancer cells. AEE788 alone or in combination with LBH589 inactivated
mitogen-activated protein kinase (MAPK) and Akt cascades. Inhibition of either MAPK and/or Akt enhanced LBH589-induced apoptosis.
In contrast, constitutively active MAPK or Akt attenuated LBH589 or LBH589 + AEE788–induced apoptosis. Increased apoptosis
was correlated with enhanced reactive oxygen species (ROS) generation. The free radical scavenger N -acetyl- l -cysteine not only substantially suppressed the ROS accumulation but also blocked the induction of apoptosis mediated by cotreatment
with AEE788 and LBH589.
Conclusion: Collectively, these results show that MAPK and Akt inactivation along with ROS generation contribute to the synergistic cytotoxicity
of the combination of AEE788 and HDAC inhibitors in a variety of human cancer cell types. This combination regimen warrants
further preclinical and possible clinical study for a broad spectrum of cancers.
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