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1.
Mol Oncol ; 17(12): 2493-2506, 2023 Dec.
Article in English | MEDLINE | ID: mdl-37872885

ABSTRACT

Leukaemia stem cells (LSCs) are the critical seed for the growth of haematological malignancies, driving the clonal expansion that enables disease initiation, relapse and often resistance. Specifically, they display inherent phenotypic and epigenetic plasticity resulting in complex heterogenic diseases. In this review, we discuss the key principles of deregulation of epigenetic processes that shape this disease evolution. We consider measures to define and quantify clonal heterogeneity, combining information from recent studies assessing mutational, transcriptional and epigenetic landscapes at single cell resolution in myeloid neoplasms (MN). We highlight the importance of integrating epigenetic and genetic information to better understand inter- and intra-patient heterogeneity and discuss how this understanding further informs evolution and progression trajectories and subsequent clinical response in MN. Under this topic, we also discuss efforts to identify mechanisms of resistance, by longitudinal analyses of patient samples. Finally, we highlight how we might target these aberrant epigenetic processes for better therapeutic outcomes and to potentially eradicate LSCs.


Subject(s)
Leukemia, Myeloid, Acute , Humans , Leukemia, Myeloid, Acute/genetics , Epigenesis, Genetic , Mutation , Stem Cells , Neoplastic Stem Cells/pathology
2.
Blood ; 141(14): 1737-1754, 2023 04 06.
Article in English | MEDLINE | ID: mdl-36577137

ABSTRACT

HOXA9 is commonly upregulated in acute myeloid leukemia (AML), in which it confers a poor prognosis. Characterizing the protein interactome of endogenous HOXA9 in human AML, we identified a chromatin complex of HOXA9 with the nuclear matrix attachment protein SAFB. SAFB perturbation phenocopied HOXA9 knockout to decrease AML proliferation, increase differentiation and apoptosis in vitro, and prolong survival in vivo. Integrated genomic, transcriptomic, and proteomic analyses further demonstrated that the HOXA9-SAFB (H9SB)-chromatin complex associates with nucleosome remodeling and histone deacetylase (NuRD) and HP1γ to repress the expression of factors associated with differentiation and apoptosis, including NOTCH1, CEBPδ, S100A8, and CDKN1A. Chemical or genetic perturbation of NuRD and HP1γ-associated catalytic activity also triggered differentiation, apoptosis, and the induction of these tumor-suppressive genes. Importantly, this mechanism is operative in other HOXA9-dependent AML genotypes. This mechanistic insight demonstrates the active HOXA9-dependent differentiation block as a potent mechanism of disease maintenance in AML that may be amenable to therapeutic intervention by targeting the H9SB interface and/or NuRD and HP1γ activity.


Subject(s)
Leukemia, Myeloid, Acute , Matrix Attachment Region Binding Proteins , Humans , Proteomics , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Leukemia, Myeloid, Acute/drug therapy , Transcription Factors/genetics , Nuclear Matrix-Associated Proteins , Chromatin , Receptors, Estrogen/genetics , Receptors, Estrogen/therapeutic use , Matrix Attachment Region Binding Proteins/genetics
3.
Nat Genet ; 53(10): 1443-1455, 2021 10.
Article in English | MEDLINE | ID: mdl-34556857

ABSTRACT

Altered transcription is a cardinal feature of acute myeloid leukemia (AML); however, exactly how mutations synergize to remodel the epigenetic landscape and rewire three-dimensional DNA topology is unknown. Here, we apply an integrated genomic approach to a murine allelic series that models the two most common mutations in AML: Flt3-ITD and Npm1c. We then deconvolute the contribution of each mutation to alterations of the epigenetic landscape and genome organization, and infer how mutations synergize in the induction of AML. Our studies demonstrate that Flt3-ITD signals to chromatin to alter the epigenetic environment and synergizes with mutations in Npm1c to alter gene expression and drive leukemia induction. These analyses also allow the identification of long-range cis-regulatory circuits, including a previously unknown superenhancer of Hoxa locus, as well as larger and more detailed gene-regulatory networks, driven by transcription factors including PU.1 and IRF8, whose importance we demonstrate through perturbation of network members.


Subject(s)
Chromatin Assembly and Disassembly/genetics , DNA, Neoplasm/chemistry , Gene Expression Regulation, Leukemic , Histones/metabolism , Leukemia, Myeloid, Acute/genetics , Mutation/genetics , Protein Processing, Post-Translational , Animals , Base Sequence , Disease Models, Animal , Enhancer Elements, Genetic/genetics , Gene Regulatory Networks , Genetic Loci , Humans , Mice, Inbred C57BL , Nuclear Proteins/metabolism , Nucleophosmin , Principal Component Analysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription, Genetic , fms-Like Tyrosine Kinase 3/metabolism
4.
Proc Natl Acad Sci U S A ; 116(24): 11997-12006, 2019 06 11.
Article in English | MEDLINE | ID: mdl-31127039

ABSTRACT

Interferons (IFNs) represent an important host defense against viruses. Type I IFNs induce JAK-STAT signaling and expression of IFN-stimulated genes (ISGs), which mediate antiviral activity. Histone deacetylases (HDACs) perform multiple functions in regulating gene expression and some class I HDACs and the class IV HDAC, HDAC11, influence type I IFN signaling. Here, HDAC4, a class II HDAC, is shown to promote type I IFN signaling and coprecipitate with STAT2. Pharmacological inhibition of class II HDAC activity, or knockout of HDAC4 from HEK-293T and HeLa cells, caused a defective response to IFN-α. This defect in HDAC4-/- cells was rescued by reintroduction of HDAC4 or catalytically inactive HDAC4, but not HDAC1 or HDAC5. ChIP analysis showed HDAC4 was recruited to ISG promoters following IFN stimulation and was needed for binding of STAT2 to these promoters. The biological importance of HDAC4 as a virus restriction factor was illustrated by the observations that (i) the replication and spread of vaccinia virus (VACV) and herpes simplex virus type 1 (HSV-1) were enhanced in HDAC4-/- cells and inhibited by overexpression of HDAC4; and (ii) HDAC4 is targeted for proteasomal degradation during VACV infection by VACV protein C6, a multifunctional IFN antagonist that coprecipitates with HDAC4 and is necessary and sufficient for HDAC4 degradation.


Subject(s)
DNA Viruses/metabolism , Histone Deacetylases/metabolism , Interferon Type I/metabolism , Repressor Proteins/metabolism , Signal Transduction/physiology , Vaccinia virus/metabolism , Vaccinia/metabolism , Viral Proteins/metabolism , Cell Line , Cell Line, Tumor , HEK293 Cells , HeLa Cells , Herpesvirus 1, Human/metabolism , Humans , Vaccinia/virology , Virus Replication/physiology
5.
Elife ; 82019 01 23.
Article in English | MEDLINE | ID: mdl-30672466

ABSTRACT

The PLZF transcription factor is essential for osteogenic differentiation of hMSCs; however, its regulation and molecular function during this process is not fully understood. Here, we revealed that the ZBTB16 locus encoding PLZF, is repressed by Polycomb (PcG) and H3K27me3 in naive hMSCs. At the pre-osteoblast stage of differentiation, the locus lost PcG binding and H3K27me3, gained JMJD3 recruitment, and H3K27ac resulting in high expression of PLZF. Subsequently, PLZF was recruited to osteogenic enhancers, influencing H3K27 acetylation and expression of nearby genes important for osteogenic function. Furthermore, we identified a latent enhancer within the ZBTB16/PLZF locus itself that became active, gained PLZF, p300 and Mediator binding and looped to the promoter of the nicotinamide N-methyltransferase (NNMT) gene. The increased expression of NNMT correlated with a decline in SAM levels, which is dependent on PLZF and is required for osteogenic differentiation.


Subject(s)
Cell Differentiation/genetics , Embryonic Development/genetics , Enhancer Elements, Genetic/genetics , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Osteogenesis/genetics , Promyelocytic Leukemia Zinc Finger Protein/metabolism , Acetylation , Cell Lineage/genetics , Chromatin/metabolism , Epigenesis, Genetic , Genetic Loci , Histones/metabolism , Humans , Lysine/metabolism , Nicotinamide N-Methyltransferase/genetics , Nicotinamide N-Methyltransferase/metabolism , Promoter Regions, Genetic , Promyelocytic Leukemia Zinc Finger Protein/genetics , Protein Binding , RNA/genetics , Transcriptome/genetics
6.
Nat Med ; 23(1): 69-78, 2017 01.
Article in English | MEDLINE | ID: mdl-27941792

ABSTRACT

In acute myeloid leukemia (AML), therapy resistance frequently occurs, leading to high mortality among patients. However, the mechanisms that render leukemic cells drug resistant remain largely undefined. Here, we identified loss of the histone methyltransferase EZH2 and subsequent reduction of histone H3K27 trimethylation as a novel pathway of acquired resistance to tyrosine kinase inhibitors (TKIs) and cytotoxic drugs in AML. Low EZH2 protein levels correlated with poor prognosis in AML patients. Suppression of EZH2 protein expression induced chemoresistance of AML cell lines and primary cells in vitro and in vivo. Low EZH2 levels resulted in derepression of HOX genes, and knockdown of HOXB7 and HOXA9 in the resistant cells was sufficient to improve sensitivity to TKIs and cytotoxic drugs. The endogenous loss of EZH2 expression in resistant cells and primary blasts from a subset of relapsed AML patients resulted from enhanced CDK1-dependent phosphorylation of EZH2 at Thr487. This interaction was stabilized by heat shock protein 90 (HSP90) and followed by proteasomal degradation of EZH2 in drug-resistant cells. Accordingly, inhibitors of HSP90, CDK1 and the proteasome prevented EZH2 degradation, decreased HOX gene expression and restored drug sensitivity. Finally, patients with reduced EZH2 levels at progression to standard therapy responded to the combination of bortezomib and cytarabine, concomitant with the re-establishment of EZH2 expression and blast clearance. These data suggest restoration of EZH2 protein as a viable approach to overcome treatment resistance in this AML patient population.


Subject(s)
Drug Resistance, Neoplasm/genetics , Enhancer of Zeste Homolog 2 Protein/genetics , Histones/metabolism , Leukemia, Myeloid, Acute/drug therapy , Protein Kinase Inhibitors , Adult , Aged , Aged, 80 and over , Animals , Antineoplastic Agents/pharmacology , Blotting, Western , Bortezomib/pharmacology , CDC2 Protein Kinase , Cell Line, Tumor , Cyclin-Dependent Kinases/metabolism , Cytarabine/pharmacology , Enhancer of Zeste Homolog 2 Protein/antagonists & inhibitors , Enhancer of Zeste Homolog 2 Protein/metabolism , Female , Flow Cytometry , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , HSP90 Heat-Shock Proteins/metabolism , Homeodomain Proteins/genetics , Humans , Immunohistochemistry , Immunoprecipitation , Indoles/pharmacology , Leukemia, Myeloid, Acute/genetics , Male , Mass Spectrometry , Mice , Middle Aged , Neoplasm Transplantation , Proteasome Endopeptidase Complex/metabolism , Protein Processing, Post-Translational , Proteomics , Pyridones/pharmacology , Young Adult
7.
Nat Struct Mol Biol ; 23(4): 349-57, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26926434

ABSTRACT

To empower experimentalists with a means for fast and comprehensive chromatin immunoprecipitation sequencing (ChIP-seq) data analyses, we introduce an integrated computational environment, EaSeq. The software combines the exploratory power of genome browsers with an extensive set of interactive and user-friendly tools for genome-wide abstraction and visualization. It enables experimentalists to easily extract information and generate hypotheses from their own data and public genome-wide datasets. For demonstration purposes, we performed meta-analyses of public Polycomb ChIP-seq data and established a new screening approach to analyze more than 900 datasets from mouse embryonic stem cells for factors potentially associated with Polycomb recruitment. EaSeq, which is freely available and works on a standard personal computer, can substantially increase the throughput of many analysis workflows, facilitate transparency and reproducibility by automatically documenting and organizing analyses, and enable a broader group of scientists to gain insights from ChIP-seq data.


Subject(s)
Chromatin Immunoprecipitation/methods , Software , Animals , Genome , Mice , Polycomb-Group Proteins/analysis
8.
Nat Commun ; 6: 6967, 2015 Apr 24.
Article in English | MEDLINE | ID: mdl-25908244

ABSTRACT

Oncogene-induced senescence (OIS) can occur in response to oncogenic insults and is considered an important tumour suppressor mechanism. Here we identify the lncRNA MIR31HG as upregulated in OIS and find that knockdown of MIR31HG promotes a strong p16(INK4A)-dependent senescence phenotype. Under normal conditions, MIR31HG is found in both nucleus and cytoplasm, but following B-RAF expression MIR31HG is located mainly in the cytoplasm. We show that MIR31HG interacts with both INK4A and MIR31HG genomic regions and with Polycomb group (PcG) proteins, and that MIR31HG is required for PcG-mediated repression of the INK4A locus. We further identify a functional enhancer, located between MIR31HG and INK4A, which becomes activated during OIS and interacts with the MIR31HG promoter. Data from melanoma patients show a negative correlation between MIR31HG and p16(INK4A) expression levels, suggesting a role for this transcript in cancer. Hence, our data provide a new lncRNA-mediated regulatory mechanism for the tumour suppressor p16(INK4A).


Subject(s)
Cellular Senescence , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Melanoma/metabolism , Proto-Oncogene Proteins B-raf/metabolism , RNA, Long Noncoding/metabolism , Animals , Enhancer Elements, Genetic , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , HEK293 Cells , Humans , Mice , Oncogenes , Phenotype , Polycomb-Group Proteins/metabolism
9.
Blood ; 121(1): 178-87, 2013 Jan 03.
Article in English | MEDLINE | ID: mdl-23152544

ABSTRACT

The origin of aberrant DNA methylation in cancer remains largely unknown. In the present study, we elucidated the DNA methylome in primary acute promyelocytic leukemia (APL) and the role of promyelocytic leukemia-retinoic acid receptor α (PML-RARα) in establishing these patterns. Cells from APL patients showed increased genome-wide DNA methylation with higher variability than healthy CD34(+) cells, promyelocytes, and remission BM cells. A core set of differentially methylated regions in APL was identified. Age at diagnosis, Sanz score, and Flt3-mutation status characterized methylation subtypes. Transcription factor-binding sites (eg, the c-myc-binding sites) were associated with low methylation. However, SUZ12- and REST-binding sites identified in embryonic stem cells were preferentially DNA hypermethylated in APL cells. Unexpectedly, PML-RARα-binding sites were also protected from aberrant DNA methylation in APL cells. Consistent with this, myeloid cells from preleukemic PML-RARα knock-in mice did not show altered DNA methylation and the expression of PML-RARα in hematopoietic progenitor cells prevented differentiation without affecting DNA methylation. Treatment of APL blasts with all-trans retinoic acid also did not result in immediate DNA methylation changes. The results of the present study suggest that aberrant DNA methylation is associated with leukemia phenotype but is not required for PML-RARα-mediated initiation of leukemogenesis.


Subject(s)
DNA Methylation , DNA, Neoplasm/genetics , Gene Expression Regulation, Leukemic , Leukemia, Promyelocytic, Acute/genetics , Transcription Factors/metabolism , Animals , Cell Transformation, Neoplastic/genetics , Chromosomes, Human/ultrastructure , CpG Islands , DNA, Neoplasm/metabolism , Disease Progression , Gene Knock-In Techniques , Hematopoietic Stem Cells/metabolism , Humans , Leukemia, Promyelocytic, Acute/drug therapy , Mice , Mice, Inbred C57BL , Neoplasm Proteins/physiology , Neoplastic Stem Cells/metabolism , Oncogene Proteins, Fusion/physiology , Phenotype , Polycomb Repressive Complex 2/metabolism , Preleukemia/genetics , Recombinant Fusion Proteins/physiology , Repressor Proteins/metabolism , Tretinoin/therapeutic use
10.
PLoS Genet ; 8(3): e1002494, 2012.
Article in English | MEDLINE | ID: mdl-22396653

ABSTRACT

Polycomb Repressive Complex (PRC) 1 and PRC2 regulate genes involved in differentiation and development. However, the mechanism for how PRC1 and PRC2 are recruited to genes in mammalian cells is unclear. Here we present evidence for an interaction between the transcription factor REST, PRC1, and PRC2 and show that RNF2 and REST co-regulate a number of neuronal genes in human teratocarcinoma cells (NT2-D1). Using NT2-D1 cells as a model of neuronal differentiation, we furthermore showed that retinoic-acid stimulation led to displacement of PRC1 at REST binding sites, reduced H3K27Me3, and increased gene expression. Genome-wide analysis of Polycomb binding in Rest⁻/⁻ and Eed⁻/⁻ mouse embryonic stem (mES) cells showed that Rest was required for PRC1 recruitment to a subset of Polycomb regulated neuronal genes. Furthermore, we found that PRC1 can be recruited to Rest binding sites independently of CpG islands and the H3K27Me3 mark. Surprisingly, PRC2 was frequently increased around Rest binding sites located in CpG-rich regions in the Rest⁻/⁻ mES cells, indicating a more complex interplay where Rest also can limit PRC2 recruitment. Therefore, we propose that Rest has context-dependent functions for PRC1- and PRC2- recruitment, which allows this transcription factor to act both as a recruiter of Polycomb as well as a limiting factor for PRC2 recruitment at CpG islands.


Subject(s)
Cell Differentiation , CpG Islands/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolism , Animals , Binding Sites , Cell Differentiation/drug effects , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Gene Expression Regulation, Developmental , HEK293 Cells , Humans , Jumonji Domain-Containing Histone Demethylases/metabolism , Mice , Neurons/metabolism , Polycomb-Group Proteins , Protein Binding , Teratoma/genetics , Tretinoin/pharmacology
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