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1.
Haematologica ; 105(9): 2286-2297, 2020 09 01.
Article in English | MEDLINE | ID: mdl-33054053

ABSTRACT

Differentiation therapies achieve remarkable success in acute promyelocytic leukemia, a subtype of acute myeloid leukemia. However, excluding acute promyelocytic leukemia, clinical benefits of differentiation therapies are negligible in acute myeloid leukemia except for mutant isocitrate dehydrogenase 1/2. Dihydroorotate dehydrogenase catalyses the fourth step of the de novo pyrimidine synthesis pathway. ASLAN003 is a highly potent dihydroorotate dehydrogenase inhibitor that induces differentiation, as well as reduces cell proliferation and viability, of acute myeloid leukemia cell lines and primary acute myeloid leukemia blasts including in chemo-resistant cells. Apoptotic pathways are triggered by ASLAN003, and it also significantly inhibits protein synthesis and activates AP-1 transcription, contributing to its differentiation promoting capacity. Finally, ASLAN003 substantially reduces leukemic burden and prolongs survival in acute myeloid leukemia xenograft mice and acute myeloid leukemia patient-derived xenograft models. Notably, the drug has no evident effect on normal hematopoietic cells and exhibits excellent safety profiles in mice, even after a prolonged period of administration. Our results, therefore, suggest that ASLAN003 is an agent targeting dihydroorotate dehydrogenase with potential in the treatment of acute myeloid leukemia. ASLAN003 is currently being evaluated in phase 2a clinical trial in acute myeloid leukemia patients.


Subject(s)
Leukemia, Myeloid, Acute , Oxidoreductases Acting on CH-CH Group Donors , Animals , Cell Differentiation , Dihydroorotate Dehydrogenase , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Mice , Oxidoreductases Acting on CH-CH Group Donors/genetics
2.
Blood ; 134(23): 2046-2058, 2019 12 05.
Article in English | MEDLINE | ID: mdl-31434700

ABSTRACT

Oncogenic EZH2 is overexpressed and extensively involved in the pathophysiology of different cancers including extranodal natural killer/T-cell lymphoma (NKTL). However, the mechanisms regarding EZH2 upregulation is poorly understood, and it still remains untargetable in NKTL. In this study, we examine EZH2 protein turnover in NKTL and identify MELK kinase as a regulator of EZH2 ubiquitination and turnover. Using quantitative mass spectrometry analysis, we observed a MELK-mediated increase of EZH2 S220 phosphorylation along with a concomitant loss of EZH2 K222 ubiquitination, suggesting a phosphorylation-dependent regulation of EZH2 ubiquitination. MELK inhibition through both chemical and genetic means led to ubiquitination and destabilization of EZH2 protein. Importantly, we determine that MELK is upregulated in NKTL, and its expression correlates with EZH2 protein expression as determined by tissue microarray derived from NKTL patients. FOXM1, which connected MELK to EZH2 signaling in glioma, was not involved in mediating EZH2 ubiquitination. Furthermore, we identify USP36 as the deubiquitinating enzyme that deubiquitinates EZH2 at K222. These findings uncover an important role of MELK and USP36 in mediating EZH2 stability in NKTL. Moreover, MELK overexpression led to decreased sensitivity to bortezomib treatment in NKTL based on deprivation of EZH2 ubiquitination. Therefore, modulation of EZH2 ubiquitination status by targeting MELK may be a new therapeutic strategy for NKTL patients with poor bortezomib response.


Subject(s)
Enhancer of Zeste Homolog 2 Protein/metabolism , Gene Expression Regulation, Neoplastic , Lymphoma, Extranodal NK-T-Cell/metabolism , Neoplasm Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Bortezomib/therapeutic use , Cell Line, Tumor , Enhancer of Zeste Homolog 2 Protein/genetics , Forkhead Box Protein M1/genetics , Forkhead Box Protein M1/metabolism , Humans , Lymphoma, Extranodal NK-T-Cell/drug therapy , Lymphoma, Extranodal NK-T-Cell/genetics , Lymphoma, Extranodal NK-T-Cell/pathology , Neoplasm Proteins/genetics , Phosphorylation/genetics , Protein Serine-Threonine Kinases/genetics , Protein Stability , Ubiquitin Thiolesterase/genetics , Ubiquitin Thiolesterase/metabolism , Ubiquitination/genetics
3.
Blood ; 128(7): 948-58, 2016 08 18.
Article in English | MEDLINE | ID: mdl-27297789

ABSTRACT

The best-understood mechanism by which EZH2 exerts its oncogenic function is through polycomb repressive complex 2 (PRC2)-mediated gene repression, which requires its histone methyltransferase activity. However, small-molecule inhibitors of EZH2 that selectively target its enzymatic activity turn out to be potent only for lymphoma cells with EZH2-activating mutation. Intriguingly, recent discoveries, including ours, have placed EZH2 into the category of transcriptional coactivators and thus raised the possibility of noncanonical signaling pathways. However, it remains unclear how EZH2 switches to this catalytic independent function. In the current study, using natural killer/T-cell lymphoma (NKTL) as a disease model, we found that phosphorylation of EZH2 by JAK3 promotes the dissociation of the PRC2 complex leading to decreased global H3K27me3 levels, while it switches EZH2 to a transcriptional activator, conferring higher proliferative capacity of the affected cells. Gene expression data analysis also suggests that the noncanonical function of EZH2 as a transcriptional activator upregulates a set of genes involved in DNA replication, cell cycle, biosynthesis, stemness, and invasiveness. Consistently, JAK3 inhibitor was able to significantly reduce the growth of NKTL cells, in an EZH2 phosphorylation-dependent manner, whereas various compounds recently developed to inhibit EZH2 methyltransferase activity have no such effect. Thus, pharmacological inhibition of JAK3 activity may provide a promising treatment option for NKTL through the novel mechanism of suppressing noncanonical EZH2 activity.


Subject(s)
Enhancer of Zeste Homolog 2 Protein/metabolism , Janus Kinase 3/metabolism , Lymphoma, T-Cell/metabolism , Natural Killer T-Cells/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Enzyme Activation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Histones/metabolism , Humans , Lymphoma, T-Cell/genetics , Lymphoma, T-Cell/pathology , Lysine/metabolism , Methylation/drug effects , Models, Biological , Natural Killer T-Cells/drug effects , Neoplasm Proteins , Phosphorylation/drug effects , Phosphotyrosine/metabolism , Polycomb Repressive Complex 2/metabolism , Protein Binding/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Subunits/metabolism , RNA Polymerase II/metabolism , Transcription Factors
4.
Br J Haematol ; 163(1): 93-103, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23888996

ABSTRACT

Acute lymphoblastic leukaemia (ALL) is the most common paediatric malignancy. Although 90% of patients are now long-term survivors, the remaining 10% have poor outcome predominantly due to drug resistance. In this study, we carried out genome-wide microRNA (miRNA) microarray analysis on diagnostic bone marrow samples to determine miRNA expression profiles associated with poor outcome in ALL. A reduced expression of MIR335 was identified as the most significant miRNA abnormality associated with poor outcome. It is well known that glucocorticoid (GC) resistance is one of the major reasons contributing to poor outcome. We show that exogenous expression of MIR335 in ALL cells increases sensitization to prednisolone-mediated apoptosis. Moreover, we demonstrate that MAPK1 is a novel target of MIR335, and that MEK/ERK inhibitor treatment enhanced prednisolone-induced cell death through the activation of BIM (BCL2L11). These results provide a possible underlying molecular mechanism to explain the association between reduced MIR335 with poor clinical outcome, and suggest that approaches to re-introduce MIR335 expression or override MAPK1 activity may offer promising therapeutic strategies in the treatment of ALL.


Subject(s)
MicroRNAs/genetics , Mitogen-Activated Protein Kinase 1/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/mortality , Cell Line, Tumor , Child , Child, Preschool , Computational Biology/methods , Drug Resistance, Neoplasm/genetics , Female , Gene Expression , Gene Expression Profiling , Gene Expression Regulation, Leukemic , Genetic Vectors/genetics , Humans , Lentivirus/genetics , MAP Kinase Signaling System/drug effects , Male , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Prednisolone/pharmacology , Prednisolone/therapeutic use , Prognosis , Recurrence
5.
Blood ; 121(22): 4512-20, 2013 May 30.
Article in English | MEDLINE | ID: mdl-23529930

ABSTRACT

The role of enhancer of zeste homolog 2 (EZH2) in cancer is complex and may vary depending on the cellular context. We found that EZH2 is aberrantly overexpressed in the majority of natural killer/T-cell lymphoma (NKTL), an aggressive lymphoid malignancy with very poor prognosis. We show that EZH2 upregulation is mediated by MYC-induced repression of its regulatory micro RNAs and EZH2 exerts oncogenic properties in NKTL. Ectopic expression of EZH2 in both primary NK cells and NKTL cell lines leads to a significant growth advantage. Conversely, knock-down of EZH2 in NKTL cell lines results in cell growth inhibition. Intriguingly, ectopic EZH2 mutant deficient for histone methyltransferase activity is also able to confer growth advantage and rescue growth inhibition on endogenous EZH2 depletion in NKTL cells, indicating an oncogenic role of EZH2 independent of its gene-silencing activity. Mechanistically, we show that EZH2 directly promotes the transcription of cyclin D1 and this effect is independent of its enzymatic activity. Furthermore, depletion of EZH2 using a PRC2 inhibitor 3-deazaneplanocin A significantly inhibits growth of NK tumor cells. Therefore, our study uncovers an oncogenic role of EZH2 independent of its methyltransferase activity in NKTL and suggests that targeting EZH2 may have therapeutic usefulness in this lymphoma.


Subject(s)
Histone-Lysine N-Methyltransferase/metabolism , Killer Cells, Natural/physiology , Lymphoma, T-Cell/physiopathology , Polycomb Repressive Complex 2/genetics , Adenosine/analogs & derivatives , Adenosine/pharmacology , Adolescent , Adult , Aged , Aged, 80 and over , Apoptosis/drug effects , Apoptosis/physiology , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin D1/genetics , Cyclin D1/metabolism , Enhancer of Zeste Homolog 2 Protein , Female , Gene Expression Regulation, Neoplastic , Histone Methyltransferases , Histone-Lysine N-Methyltransferase/genetics , Humans , Killer Cells, Natural/cytology , Lymphoma, T-Cell/pathology , Male , MicroRNAs/metabolism , Middle Aged , Mutagenesis/physiology , Polycomb Repressive Complex 2/metabolism , Promoter Regions, Genetic/physiology , Up-Regulation/physiology , Young Adult
6.
Blood ; 118(18): 4919-29, 2011 Nov 03.
Article in English | MEDLINE | ID: mdl-21921041

ABSTRACT

We performed a comprehensive genome-wide miRNA expression profiling of extranodal nasal-type natural killer/T-cell lymphoma (NKTL) using formalin-fixed paraffin-embedded tissue (n = 30) and NK cell lines (n = 6) compared with normal NK cells, with the objective of understanding the pathogenetic role of miRNA deregulation in NKTL. Compared with normal NK cells, differentially expressed miRNAs in NKTL are predominantly down-regulated. Re-expression of down-regulated miRNAs, such as miR-101, miR-26a, miR26b, miR-28-5, and miR-363, reduced the growth of the NK cell line and modulated the expression of their predicted target genes, suggesting the potential functional role of the deregulated miRNAs in the oncogenesis of NKTL. Taken together, the predicted targets whose expression is inversely correlated with the expression of deregulated miRNA in NKTL are significantly enriched for genes involved in cell cycle-related, p53, and MAPK signaling pathways. We also performed immunohistochemical validation for selected target proteins and found overexpression of MUM1, BLIMP1, and STMN1 in NKTL, and notably, a corresponding increase in MYC expression. Because MYC is known to cause repression of miRNA expression, it is possible that MYC activation in NKTL may contribute to the suppression of the miRNAs regulating MUM1, BLIMP1, and STMN1.


Subject(s)
Lymphoma, Extranodal NK-T-Cell/genetics , MicroRNAs/genetics , Animals , Caenorhabditis elegans/genetics , Cell Line, Tumor , Cluster Analysis , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/physiology , Humans , Killer Cells, Natural/metabolism , Killer Cells, Natural/pathology , Lymphoma, Extranodal NK-T-Cell/diagnosis , Lymphoma, Extranodal NK-T-Cell/metabolism , Lymphoma, Extranodal NK-T-Cell/therapy , MicroRNAs/metabolism , Microarray Analysis , Molecular Targeted Therapy , Prognosis , Signal Transduction/genetics , Signal Transduction/physiology , Transfection
7.
J Cell Sci ; 122(Pt 22): 4150-9, 2009 Nov 15.
Article in English | MEDLINE | ID: mdl-19861492

ABSTRACT

Stat3 is a member of the signal transducer and activator of transcription family, which is important for cytokine signaling as well as for a number of cellular processes including cell proliferation, anti-apoptosis and immune responses. In recent years, evidence has emerged suggesting that Stat3 also participates in cell invasion and motility. However, how Stat3 regulates these processes remains poorly understood. Here, we find that loss of Stat3 expression in mouse embryonic fibroblasts leads to an elevation of Rac1 activity, which promotes a random mode of migration by reducing directional persistence and formation of actin stress fibers. Through rescue experiments, we demonstrate that Stat3 can regulate the activation of Rac1 to mediate persistent directional migration and that this function is not dependent on Stat3 transcriptional activity. We find that Stat3 binds to betaPIX, a Rac1 activator, and that this interaction could represent a mechanism by which cytoplasmic Stat3 regulates Rac1 activity to modulate the organization of actin cytoskeleton and directional migration.


Subject(s)
Cell Movement/physiology , Guanine Nucleotide Exchange Factors/metabolism , STAT3 Transcription Factor/metabolism , Stress Fibers/metabolism , rac1 GTP-Binding Protein/metabolism , Actins , Animals , Cells, Cultured , Cytoplasm/metabolism , Fibroblasts/cytology , Fibroblasts/physiology , Gene Knockdown Techniques , Gene Knockout Techniques , Guanine Nucleotide Exchange Factors/genetics , Humans , Mice , Rho Guanine Nucleotide Exchange Factors , STAT3 Transcription Factor/genetics , Transcriptional Activation , rac1 GTP-Binding Protein/genetics
8.
J Biol Chem ; 280(13): 12747-57, 2005 Apr 01.
Article in English | MEDLINE | ID: mdl-15677447

ABSTRACT

The interleukin (IL)-6-type cytokines play major roles in a variety of biological processes by signaling through a common receptor subunit, glycoprotein (gp) 130. We performed yeast two-hybrid screening to identify new binding partners of the activated gp130 and the associated Janus kinases. LMO4, a LIM domain-containing protein that belongs to a family of oncogenes, was identified in this assay. Further studies show that LMO4 associates with gp130 and Janus kinase1 in several mammalian cell types. It also interacts with protein-tyrosine phosphatase 2 (SHP2) and suppressor of cytokine signaling 3 (SOCS3). The binding domains involved in these interactions were mapped, and the interactions were shown to be in a direct manner by in vitro binding assays. It is likely that LMO4 exists in the gp130 complex. The cellular localization of LMO4 was detected primarily in the nucleus with a substantial amount also detected in the cytoplasm in several cell types. The effect of LMO4 in IL-6 signaling was subsequently examined. Overexpression of LMO4 enhanced the transcriptional activity and target gene expression of Stat 3 (signal transducers and activators of transcription 3). Consistent with this, silencing LMO4 expression in stable cell lines expressing the small interfering RNA of LMO4 decreased Stat3 activity. Furthermore, the half-life of gp130 was shortened, and the production of acute phase proteins induced by IL-6 was reduced. Together, our data reveal a positive regulatory role of LMO4 in IL-6 signaling, possibly by acting as a scaffold for stabilization of the gp130 complex. These studies may open up a link between the oncogenic effect of LMO proteins and their regulatory role in cytokine signaling in general.


Subject(s)
Antigens, CD/chemistry , Antigens, CD/physiology , Homeodomain Proteins/physiology , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/physiology , Signal Transduction , Transcription Factors/physiology , Adaptor Proteins, Signal Transducing , Animals , Blotting, Northern , COS Cells , Cell Line , Cell Line, Tumor , Cell Nucleus/metabolism , Cytokine Receptor gp130 , Cytokines/metabolism , Cytoplasm/metabolism , DNA-Binding Proteins/metabolism , Down-Regulation , Gene Silencing , Genes, Reporter , Glutathione Transferase/metabolism , Homeodomain Proteins/chemistry , Humans , Immunoprecipitation , Intracellular Signaling Peptides and Proteins , LIM Domain Proteins , Microscopy, Fluorescence , Phosphorylation , Plasmids/metabolism , Protein Binding , Protein Phosphatase 2 , Protein Structure, Tertiary , Protein Tyrosine Phosphatase, Non-Receptor Type 11 , Protein Tyrosine Phosphatases/chemistry , RNA, Small Interfering/metabolism , STAT3 Transcription Factor , Trans-Activators/metabolism , Transcription Factors/chemistry , Transfection , Two-Hybrid System Techniques , Tyrosine/chemistry , Tyrosine/metabolism
9.
Mol Biol Cell ; 16(4): 1569-83, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15659653

ABSTRACT

Islet1 (Isl1) belongs to the LIM homeodomain transcription factor family. Its roles in differentiation of motor neurons and organogenesis of pancreas and heart have been revealed. However, less is known about its regulatory mechanism and the target genes. In this study, we identified interactions between Isl1 and Janus tyrosine kinase (JAK), as well as signal transducer and activator of transcription (Stat)3, but not Stat1 and Stat5, in mammalian cells. We found that Isl1 not only forms a complex with Jak1 and Stat3 but also triggers the tyrosine phosphorylation of Jak1 and its kinase activity, thereby elevating the tyrosine phosphorylation, DNA binding activity, and target gene expression of Stat3. In vivo, the tyrosine-phosphorylated Stat3 was colocalized with Isl1 in the nucleus of the mouse motor neurons in spinal cord after nerve injury. Correspondingly, electroporation of Isl1 and Stat3 into the neural tube of chick embryos resulted in the activation of a reporter gene expression controlled by a Stat3 regulatory sequence, and cotransfection of Isl1 and Stat3 promoted the proliferation of the mouse motor neuron cells. Our data suggest a novel role of Isl1 as an adaptor for Jak1 and Stat3 and reveal a possible functional link between LIM homeodomain transcription factors and the Jak-Stat pathway.


Subject(s)
DNA-Binding Proteins/metabolism , Homeodomain Proteins/metabolism , Nerve Tissue Proteins/metabolism , Protein-Tyrosine Kinases/metabolism , Trans-Activators/metabolism , Animals , Cell Line , Cell Nucleus/metabolism , Cell Proliferation , Chick Embryo , Chlorocebus aethiops , DNA/metabolism , DNA-Binding Proteins/genetics , Gene Expression Regulation , Genes, Reporter/genetics , Homeodomain Proteins/genetics , Humans , Janus Kinase 1 , LIM-Homeodomain Proteins , Mice , Motor Neurons/cytology , Motor Neurons/metabolism , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Mutation/genetics , Nerve Tissue Proteins/genetics , Phosphorylation , Phosphotyrosine/metabolism , Protein Binding , Rats , STAT3 Transcription Factor , Trans-Activators/genetics , Transcription Factors , Transcription, Genetic/genetics
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