Publications by authors named "Thomas A Milne"

56 Publications

Defining genome architecture at base-pair resolution.

Nature 2021 07 9;595(7865):125-129. Epub 2021 Jun 9.

MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.

In higher eukaryotes, many genes are regulated by enhancers that are 10-10 base pairs (bp) away from the promoter. Enhancers contain transcription-factor-binding sites (which are typically around 7-22 bp), and physical contact between the promoters and enhancers is thought to be required to modulate gene expression. Although chromatin architecture has been mapped extensively at resolutions of 1 kilobase and above; it has not been possible to define physical contacts at the scale of the proteins that determine gene expression. Here we define these interactions in detail using a chromosome conformation capture method (Micro-Capture-C) that enables the physical contacts between different classes of regulatory elements to be determined at base-pair resolution. We find that highly punctate contacts occur between enhancers, promoters and CCCTC-binding factor (CTCF) sites and we show that transcription factors have an important role in the maintenance of the contacts between enhancers and promoters. Our data show that interactions between CTCF sites are increased when active promoters and enhancers are located within the intervening chromatin. This supports a model in which chromatin loop extrusion is dependent on cohesin loading at active promoters and enhancers, which explains the formation of tissue-specific chromatin domains without changes in CTCF binding.
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http://dx.doi.org/10.1038/s41586-021-03639-4DOI Listing
July 2021

A KMT2A-AFF1 gene regulatory network highlights the role of core transcription factors and reveals the regulatory logic of key downstream target genes.

Genome Res 2021 Jun 4. Epub 2021 Jun 4.

MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, OX3 9DS, United Kingdom.

Regulatory interactions mediated by transcription factors (TFs) make up complex networks that control cellular behavior. Fully understanding these gene regulatory networks (GRNs) offers greater insight into the consequences of disease-causing perturbations than can be achieved by studying single TF binding events in isolation. Chromosomal translocations of the () gene produce KMT2A fusion proteins such as KMT2A-AFF1 (previously MLL-AF4), causing poor prognosis acute lymphoblastic leukemias (ALLs) that sometimes relapse as acute myeloid leukemias (AMLs). KMT2A-AFF1 drives leukemogenesis through direct binding and inducing the aberrant overexpression of key genes, such as the anti-apoptotic factor and the proto-oncogene However, studying direct binding alone does not incorporate possible network-generated regulatory outputs, including the indirect induction of gene repression. To better understand the KMT2A-AFF1-driven regulatory landscape, we integrated ChIP-seq, patient RNA-seq, and CRISPR essentiality screens to generate a model GRN. This GRN identified several key transcription factors such as RUNX1 that regulate target genes downstream of KMT2A-AFF1 using feed-forward loop (FFL) and cascade motifs. A core set of nodes are present in both ALL and AML, and CRISPR screening revealed several factors that help mediate response to the drug venetoclax. Using our GRN, we then identified a KMT2A-AFF1:RUNX1 cascade that represses , as well as KMT2A-AFF1-driven FFLs that regulate and through combinatorial TF activity. This illustrates how our GRN can be used to better connect KMT2A-AFF1 behavior to downstream pathways that contribute to leukemogenesis, and potentially predict shifts in gene expression that mediate drug response.
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http://dx.doi.org/10.1101/gr.268490.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8256865PMC
June 2021

Chromatin accessibility governs the differential response of cancer and T cells to arginine starvation.

Cell Rep 2021 May;35(6):109101

MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK.

Depleting the microenvironment of important nutrients such as arginine is a key strategy for immune evasion by cancer cells. Many tumors overexpress arginase, but it is unclear how these cancers, but not T cells, tolerate arginine depletion. In this study, we show that tumor cells synthesize arginine from citrulline by upregulating argininosuccinate synthetase 1 (ASS1). Under arginine starvation, ASS1 transcription is induced by ATF4 and CEBPβ binding to an enhancer within ASS1. T cells cannot induce ASS1, despite the presence of active ATF4 and CEBPβ, as the gene is repressed. Arginine starvation drives global chromatin compaction and repressive histone methylation, which disrupts ATF4/CEBPβ binding and target gene transcription. We find that T cell activation is impaired in arginine-depleted conditions, with significant metabolic perturbation linked to incomplete chromatin remodeling and misregulation of key genes. Our results highlight a T cell behavior mediated by nutritional stress, exploited by cancer cells to enable pathological immune evasion.
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http://dx.doi.org/10.1016/j.celrep.2021.109101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8131582PMC
May 2021

BET inhibition disrupts transcription but retains enhancer-promoter contact.

Nat Commun 2021 01 11;12(1):223. Epub 2021 Jan 11.

MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, NIHR Oxford Biomedical Research Centre Haematology Theme, Radcliffe Department of Medicine, University of Oxford, Oxford, OX3 9DS, UK.

Enhancers are DNA sequences that enable complex temporal and tissue-specific regulation of genes in higher eukaryotes. Although it is not entirely clear how enhancer-promoter interactions can increase gene expression, this proximity has been observed in multiple systems at multiple loci and is thought to be essential for the maintenance of gene expression. Bromodomain and Extra-Terminal domain (BET) and Mediator proteins have been shown capable of forming phase condensates and are thought to be essential for super-enhancer function. Here, we show that targeting of cells with inhibitors of BET proteins or pharmacological degradation of BET protein Bromodomain-containing protein 4 (BRD4) has a strong impact on transcription but very little impact on enhancer-promoter interactions. Dissolving phase condensates reduces BRD4 and Mediator binding at enhancers and can also strongly affect gene transcription, without disrupting enhancer-promoter interactions. These results suggest that activation of transcription and maintenance of enhancer-promoter interactions are separable events. Our findings further indicate that enhancer-promoter interactions are not dependent on high levels of BRD4 and Mediator, and are likely maintained by a complex set of factors including additional activator complexes and, at some sites, CTCF and cohesin.
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http://dx.doi.org/10.1038/s41467-020-20400-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7801379PMC
January 2021

The MLL family of proteins in normal development and disease.

Authors:
Thomas A Milne

Biochim Biophys Acta Gene Regul Mech 2020 12 4;1863(12):194648. Epub 2020 Nov 4.

MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, NIHR Oxford Biomedical Research Centre Haematology Theme, Radcliffe Department of Medicine, University of Oxford, Oxford, UK. Electronic address:

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http://dx.doi.org/10.1016/j.bbagrm.2020.194648DOI Listing
December 2020

The BET inhibitor CPI203 promotes ex vivo expansion of cord blood long-term repopulating HSCs and megakaryocytes.

Blood 2020 11;136(21):2410-2415

Nuffield Division of Clinical Laboratory Medicine, Radcliffe Department of Medicine, University of Oxford.

Although cytokine-mediated expansion of human hematopoietic stem cells (HSCs) can result in high yields of hematopoietic progenitor cells, this generally occurs at the expense of reduced bone marrow HSC repopulating ability, thereby limiting potential therapeutic applications. Because bromodomain-containing proteins (BCPs) have been demonstrated to regulate mouse HSC self-renewal and stemness, we screened small molecules targeting various BCPs as potential agents for ex vivo expansion of human HSCs. Of 10 compounds tested, only the bromodomain and extra-terminal motif inhibitor CPI203 enhanced the expansion of human cord blood HSCs without losing cell viability in vitro. The expanded cells also demonstrated improved engraftment and repopulation in serial transplantation assays. Transcriptomic and functional studies showed that the expansion of long-term repopulating HSCs was accompanied by synchronized expansion and maturation of megakaryocytes consistent with CPI203-mediated reprogramming of cord blood hematopoietic stem and progenitor cells. This approach may therefore prove beneficial for ex vivo gene editing, for enhanced platelet production, and for the improved usage of cord blood for transplantation research and therapy.
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http://dx.doi.org/10.1182/blood.2020005357DOI Listing
November 2020

H3K79me2/3 controls enhancer-promoter interactions and activation of the pan-cancer stem cell marker PROM1/CD133 in MLL-AF4 leukemia cells.

Leukemia 2021 01 2;35(1):90-106. Epub 2020 Apr 2.

MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, NIHR Oxford Biomedical Research Centre Haematology Theme, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.

MLL gene rearrangements (MLLr) are a common cause of aggressive, incurable acute lymphoblastic leukemias (ALL) in infants and children, most of which originate in utero. The most common MLLr produces an MLL-AF4 fusion protein. MLL-AF4 promotes leukemogenesis by activating key target genes, mainly through recruitment of DOT1L and increased histone H3 lysine-79 methylation (H3K79me2/3). One key MLL-AF4 target gene is PROM1, which encodes CD133 (Prominin-1). CD133 is a pentaspan transmembrane glycoprotein that represents a potential pan-cancer target as it is found on multiple cancer stem cells. Here we demonstrate that aberrant PROM1/CD133 expression is essential for leukemic cell growth, mediated by direct binding of MLL-AF4. Activation is controlled by an intragenic H3K79me2/3 enhancer element (KEE) leading to increased enhancer-promoter interactions between PROM1 and the nearby gene TAPT1. This dual locus regulation is reflected in a strong correlation of expression in leukemia. We find that in PROM1/CD133 non-expressing cells, the PROM1 locus is repressed by polycomb repressive complex 2 (PRC2) binding, associated with reduced expression of TAPT1, partially due to loss of interactions with the PROM1 locus. Together, these results provide the first detailed analysis of PROM1/CD133 regulation that explains CD133 expression in MLLr ALL.
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http://dx.doi.org/10.1038/s41375-020-0808-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7787973PMC
January 2021

Discovery of a CD10-negative B-progenitor in human fetal life identifies unique ontogeny-related developmental programs.

Blood 2019 09 5;134(13):1059-1071. Epub 2019 Aug 5.

Department of Paediatrics and.

Human lymphopoiesis is a dynamic lifelong process that starts in utero 6 weeks postconception. Although fetal B-lymphopoiesis remains poorly defined, it is key to understanding leukemia initiation in early life. Here, we provide a comprehensive analysis of the human fetal B-cell developmental hierarchy. We report the presence in fetal tissues of 2 distinct CD19 B-progenitors, an adult-type CD10+ve ProB-progenitor and a new CD10-ve PreProB-progenitor, and describe their molecular and functional characteristics. PreProB-progenitors and ProB-progenitors appear early in the first trimester in embryonic liver, followed by a sustained second wave of B-progenitor development in fetal bone marrow (BM), where together they form >40% of the total hematopoietic stem cell/progenitor pool. Almost one-third of fetal B-progenitors are CD10-ve PreProB-progenitors, whereas, by contrast, PreProB-progenitors are almost undetectable (0.53% ± 0.24%) in adult BM. Single-cell transcriptomics and functional assays place fetal PreProB-progenitors upstream of ProB-progenitors, identifying them as the first B-lymphoid-restricted progenitor in human fetal life. Although fetal BM PreProB-progenitors and ProB-progenitors both give rise solely to B-lineage cells, they are transcriptionally distinct. As with their fetal counterparts, adult BM PreProB-progenitors give rise only to B-lineage cells in vitro and express the expected B-lineage gene expression program. However, fetal PreProB-progenitors display a distinct, ontogeny-related gene expression pattern that is not seen in adult PreProB-progenitors, and they share transcriptomic signatures with CD10-ve B-progenitor infant acute lymphoblastic leukemia blast cells. These data identify PreProB-progenitors as the earliest B-lymphoid-restricted progenitor in human fetal life and suggest that this fetal-restricted committed B-progenitor might provide a permissive cellular context for prenatal B-progenitor leukemia initiation.
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http://dx.doi.org/10.1182/blood.2019001289DOI Listing
September 2019

DOT1L inhibition reveals a distinct subset of enhancers dependent on H3K79 methylation.

Nat Commun 2019 06 26;10(1):2803. Epub 2019 Jun 26.

MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, NIHR Oxford Biomedical Research Centre Haematology Theme, Radcliffe Department of Medicine, University of Oxford, Oxford, OX3 9DS, UK.

Enhancer elements are a key regulatory feature of many important genes. Several general features including the presence of specific histone modifications are used to demarcate potentially active enhancers. Here we reveal that putative enhancers marked with H3 lysine 79 (H3K79) di or trimethylation (me2/3) (which we name H3K79me2/3 enhancer elements or KEEs) can be found in multiple cell types. Mixed lineage leukemia gene (MLL) rearrangements (MLL-r) such as MLL-AF4 are a major cause of incurable acute lymphoblastic leukemias (ALL). Using the DOT1L inhibitor EPZ-5676 in MLL-AF4 leukemia cells, we show that H3K79me2/3 is required for maintaining chromatin accessibility, histone acetylation and transcription factor binding specifically at KEEs but not non-KEE enhancers. We go on to show that H3K79me2/3 is essential for maintaining enhancer-promoter interactions at a subset of KEEs. Together, these data implicate H3K79me2/3 as having a functional role at a subset of active enhancers in MLL-AF4 leukemia cells.
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http://dx.doi.org/10.1038/s41467-019-10844-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594956PMC
June 2019

Addendum: Precise tuning of gene expression levels in mammalian cells.

Nat Commun 2019 06 10;10(1):2622. Epub 2019 Jun 10.

Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, OX3 9DS, UK.

Following re-sequencing of the miSFIT constructs used in the paper, two of the construct variants inserted into the 3'UTR of PD-1, namely '12C' and '17A, 18G', have been found to contain additional insertions not present in the other construct variants. The data points corresponding to these constructs in Figs. 2c, f and Supplementary Fig. 9 are therefore no longer valid. However the overall conclusion that step-wise control over gene expression levels using the miSFIT constructs remains unaffected by these errors. Updated versions of Fig. 2 and Supplementary Fig. 9 are presented in the accompanying Addendum.
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http://dx.doi.org/10.1038/s41467-019-10615-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6557827PMC
June 2019

Why are so many MLL lysine methyltransferases required for normal mammalian development?

Cell Mol Life Sci 2019 Aug 16;76(15):2885-2898. Epub 2019 May 16.

MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, NIHR Oxford Biomedical Research Centre Haematology Theme, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.

The mixed lineage leukemia (MLL) family of proteins became known initially for the leukemia link of its founding member. Over the decades, the MLL family has been recognized as an important class of histone H3 lysine 4 (H3K4) methyltransferases that control key aspects of normal cell physiology and development. Here, we provide a brief history of the discovery and study of this family of proteins. We address two main questions: why are there so many H3K4 methyltransferases in mammals; and is H3K4 methylation their key function?
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http://dx.doi.org/10.1007/s00018-019-03143-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6647185PMC
August 2019

Decoupling tRNA promoter and processing activities enables specific Pol-II Cas9 guide RNA expression.

Nat Commun 2019 04 2;10(1):1490. Epub 2019 Apr 2.

Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, OX3 9DS, UK.

Spatial/temporal control of Cas9 guide RNA expression could considerably expand the utility of CRISPR-based technologies. Current approaches based on tRNA processing offer a promising strategy but suffer from high background. Here, to address this limitation, we present a screening platform which allows simultaneous measurements of the promoter strength, 5', and 3' processing efficiencies across a library of tRNA variants. This analysis reveals that the sequence determinants underlying these activities, while overlapping, are dissociable. Rational design based on the ensuing principles allowed us to engineer an improved tRNA scaffold that enables highly specific guide RNA production from a Pol-II promoter. When benchmarked against other reported systems this tRNA scaffold is superior to most alternatives, and is equivalent in function to an optimized version of the Csy4-based guide RNA release system. The results and methods described in this manuscript enable avenues of research both in genome engineering and basic tRNA biology.
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http://dx.doi.org/10.1038/s41467-019-09148-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6445147PMC
April 2019

Precise tuning of gene expression levels in mammalian cells.

Nat Commun 2019 02 18;10(1):818. Epub 2019 Feb 18.

Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, OX3 9DS, UK.

Precise, analogue regulation of gene expression is critical for cellular function in mammals. In contrast, widely employed experimental and therapeutic approaches such as knock-in/out strategies are more suitable for binary control of gene activity. Here we report on a method for precise control of gene expression levels in mammalian cells using engineered microRNA response elements (MREs). First, we measure the efficacy of thousands of synthetic MRE variants under the control of an endogenous microRNA by high-throughput sequencing. Guided by this data, we establish a library of microRNA silencing-mediated fine-tuners (miSFITs) of varying strength that can be employed to precisely control the expression of user-specified genes. We apply this technology to tune the T-cell co-inhibitory receptor PD-1 and to explore how antigen expression influences T-cell activation and tumour growth. Finally, we employ CRISPR/Cas9 mediated homology directed repair to introduce miSFITs into the BRCA1 3'UTR, demonstrating that this versatile tool can be used to tune endogenous genes.
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http://dx.doi.org/10.1038/s41467-019-08777-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379387PMC
February 2019

Author Correction: Metabolic gatekeeper function of B-lymphoid transcription factors.

Nature 2018 06;558(7711):E5

Department of Systems Biology, Beckman Research Institute and City of Hope National Medical Center, Pasadena, California, 91016, USA.

In Fig. 3c of this Letter, the the effects of CRISPR-Cas9-mediated deletion of NR3C1, TXNIP and CNR2 in patient-derived B-lineage leukaemia cells were shown. For curves depicting NR3C1 (left graph), data s for TXNIP (middle graph) were inadvertently plotted. This figure has been corrected online, and the original Fig. 3c is shown as Supplementary Information to this Amendment for transparency. The error does not affect the conclusions of the Letter. In addition, Source Data files have been added for the Figs. 1-4 and Extended Data Figs. 1-10 of the original Letter.
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http://dx.doi.org/10.1038/s41586-018-0164-5DOI Listing
June 2018

The basic helix-loop-helix transcription factor SHARP1 is an oncogenic driver in MLL-AF6 acute myelogenous leukemia.

Nat Commun 2018 04 24;9(1):1622. Epub 2018 Apr 24.

Cancer Science Institute of Singapore, National University of Singapore, Singapore, 117599, Singapore.

Acute Myeloid Leukemia (AML) with MLL gene rearrangements demonstrate unique gene expression profiles driven by MLL-fusion proteins. Here, we identify the circadian clock transcription factor SHARP1 as a novel oncogenic target in MLL-AF6 AML, which has the worst prognosis among all subtypes of MLL-rearranged AMLs. SHARP1 is expressed solely in MLL-AF6 AML, and its expression is regulated directly by MLL-AF6/DOT1L. Suppression of SHARP1 induces robust apoptosis of human MLL-AF6 AML cells. Genetic deletion in mice delays the development of leukemia and attenuated leukemia-initiating potential, while sparing normal hematopoiesis. Mechanistically, SHARP1 binds to transcriptionally active chromatin across the genome and activates genes critical for cell survival as well as key oncogenic targets of MLL-AF6. Our findings demonstrate the unique oncogenic role for SHARP1 in MLL-AF6 AML.
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http://dx.doi.org/10.1038/s41467-018-03854-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5915391PMC
April 2018

Ezh2 and Runx1 Mutations Collaborate to Initiate Lympho-Myeloid Leukemia in Early Thymic Progenitors.

Cancer Cell 2018 02;33(2):274-291.e8

Haematopoietic Stem Cell Biology Laboratory, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 9DS, UK; MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 9DS, UK; NIHR Biomedical Research Centre, Churchill Hospital, Oxford OX3 7LE, UK. Electronic address:

Lympho-myeloid restricted early thymic progenitors (ETPs) are postulated to be the cell of origin for ETP leukemias, a therapy-resistant leukemia associated with frequent co-occurrence of EZH2 and RUNX1 inactivating mutations, and constitutively activating signaling pathway mutations. In a mouse model, we demonstrate that Ezh2 and Runx1 inactivation targeted to early lymphoid progenitors causes a marked expansion of pre-leukemic ETPs, showing transcriptional signatures characteristic of ETP leukemia. Addition of a RAS-signaling pathway mutation (Flt3-ITD) results in an aggressive leukemia co-expressing myeloid and lymphoid genes, which can be established and propagated in vivo by the expanded ETPs. Both mouse and human ETP leukemias show sensitivity to BET inhibition in vitro and in vivo, which reverses aberrant gene expression induced by Ezh2 inactivation.
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http://dx.doi.org/10.1016/j.ccell.2018.01.006DOI Listing
February 2018

LEDGF: a leukemia-specific target.

Authors:
Thomas A Milne

Blood 2018 01;131(1):4-5

University of Oxford.

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http://dx.doi.org/10.1182/blood-2017-11-815449DOI Listing
January 2018

In situ functional dissection of RNA cis-regulatory elements by multiplex CRISPR-Cas9 genome engineering.

Nat Commun 2017 12 13;8(1):2109. Epub 2017 Dec 13.

Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, OX3 9DS, UK.

RNA regulatory elements (RREs) are an important yet relatively under-explored facet of gene regulation. Deciphering the prevalence and functional impact of this post-transcriptional control layer requires technologies for disrupting RREs without perturbing cellular homeostasis. Here we describe genome-engineering based evaluation of RNA regulatory element activity (GenERA), a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 platform for in situ high-content functional analysis of RREs. We use GenERA to survey the entire regulatory landscape of a 3'UTR, and apply it in a multiplex fashion to analyse combinatorial interactions between sets of miRNA response elements (MREs), providing strong evidence for cooperative activity. We also employ this technology to probe the functionality of an entire MRE network under cellular homeostasis, and show that high-resolution analysis of the GenERA dataset can be used to extract functional features of MREs. This study provides a genome editing-based multiplex strategy for direct functional interrogation of RNA cis-regulatory elements in a native cellular environment.
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http://dx.doi.org/10.1038/s41467-017-00686-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5727397PMC
December 2017

The Cks1/Cks2 axis fine-tunes Mll1 expression and is crucial for MLL-rearranged leukaemia cell viability.

Biochim Biophys Acta Mol Cell Res 2018 Jan 20;1865(1):105-116. Epub 2017 Sep 20.

Department of Medical and Molecular Genetics, King's College London, Faculty of Life Sciences and Medicine, London, UK.

The Cdc28 protein kinase subunits, Cks1 and Cks2, play dual roles in Cdk-substrate specificity and Cdk-independent protein degradation, in concert with the E3 ubiquitin ligase complexes SCF and APC. Notable targets controlled by Cks include p27 and Cyclin A. Here, we demonstrate that Cks1 and Cks2 proteins interact with both the Mll and Mll subunits of Mll1 (Mixed-lineage leukaemia 1), and together, the Cks proteins define Mll1 levels throughout the cell cycle. Overexpression of CKS1B and CKS2 is observed in multiple human cancers, including various MLL-rearranged (MLLr) AML subtypes. To explore the importance of MLL-Fusion Protein regulation by CKS1/2, we used small molecule inhibitors (MLN4924 and C1) to modulate their protein degradation functions. These inhibitors specifically reduced the proliferation of MLLr cell lines compared to primary controls. Altogether, this study uncovers a novel regulatory pathway for MLL1, which may open a new therapeutic approach to MLLr leukaemia.
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http://dx.doi.org/10.1016/j.bbamcr.2017.09.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5701546PMC
January 2018

Hepcidin is regulated by promoter-associated histone acetylation and HDAC3.

Nat Commun 2017 09 1;8(1):403. Epub 2017 Sep 1.

MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, OX3 9DS, UK.

Hepcidin regulates systemic iron homeostasis. Suppression of hepcidin expression occurs physiologically in iron deficiency and increased erythropoiesis but is pathologic in thalassemia and hemochromatosis. Here we show that epigenetic events govern hepcidin expression. Erythropoiesis and iron deficiency suppress hepcidin via erythroferrone-dependent and -independent mechanisms, respectively, in vivo, but both involve reversible loss of H3K9ac and H3K4me3 at the hepcidin locus. In vitro, pan-histone deacetylase inhibition elevates hepcidin expression, and in vivo maintains H3K9ac at hepcidin-associated chromatin and abrogates hepcidin suppression by erythropoietin, iron deficiency, thalassemia, and hemochromatosis. Histone deacetylase 3 and its cofactor NCOR1 regulate hepcidin; histone deacetylase 3 binds chromatin at the hepcidin locus, and histone deacetylase 3 knockdown counteracts hepcidin suppression induced either by erythroferrone or by inhibiting bone morphogenetic protein signaling. In iron deficient mice, the histone deacetylase 3 inhibitor RGFP966 increases hepcidin, and RNA sequencing confirms hepcidin is one of the genes most differentially regulated by this drug in vivo. We conclude that suppression of hepcidin expression involves epigenetic regulation by histone deacetylase 3.Hepcidin controls systemic iron levels by inhibiting intestinal iron absorption and iron recycling. Here, Pasricha et al. demonstrate that the hepcidin-chromatin locus displays HDAC3-mediated reversible epigenetic modifications during both erythropoiesis and iron deficiency.
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http://dx.doi.org/10.1038/s41467-017-00500-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5581335PMC
September 2017

Metabolic gatekeeper function of B-lymphoid transcription factors.

Nature 2017 02 13;542(7642):479-483. Epub 2017 Feb 13.

Department of Systems Biology, Beckman Research Institute and City of Hope National Medical Center, Pasadena, California 91016, USA.

B-lymphoid transcription factors, such as PAX5 and IKZF1, are critical for early B-cell development, yet lesions of the genes encoding these transcription factors occur in over 80% of cases of pre-B-cell acute lymphoblastic leukaemia (ALL). The importance of these lesions in ALL has, until now, remained unclear. Here, by combining studies using chromatin immunoprecipitation with sequencing and RNA sequencing, we identify a novel B-lymphoid program for transcriptional repression of glucose and energy supply. Our metabolic analyses revealed that PAX5 and IKZF1 enforce a state of chronic energy deprivation, resulting in constitutive activation of the energy-stress sensor AMPK. Dominant-negative mutants of PAX5 and IKZF1, however, relieved this glucose and energy restriction. In a transgenic pre-B ALL mouse model, the heterozygous deletion of Pax5 increased glucose uptake and ATP levels by more than 25-fold. Reconstitution of PAX5 and IKZF1 in samples from patients with pre-B ALL restored a non-permissive state and induced energy crisis and cell death. A CRISPR/Cas9-based screen of PAX5 and IKZF1 transcriptional targets identified the products of NR3C1 (encoding the glucocorticoid receptor), TXNIP (encoding a glucose-feedback sensor) and CNR2 (encoding a cannabinoid receptor) as central effectors of B-lymphoid restriction of glucose and energy supply. Notably, transport-independent lipophilic methyl-conjugates of pyruvate and tricarboxylic acid cycle metabolites bypassed the gatekeeper function of PAX5 and IKZF1 and readily enabled leukaemic transformation. Conversely, pharmacological TXNIP and CNR2 agonists and a small-molecule AMPK inhibitor strongly synergized with glucocorticoids, identifying TXNIP, CNR2 and AMPK as potential therapeutic targets. Furthermore, our results provide a mechanistic explanation for the empirical finding that glucocorticoids are effective in the treatment of B-lymphoid but not myeloid malignancies. Thus, B-lymphoid transcription factors function as metabolic gatekeepers by limiting the amount of cellular ATP to levels that are insufficient for malignant transformation.
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http://dx.doi.org/10.1038/nature21076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5621518PMC
February 2017

Mouse models of MLL leukemia: recapitulating the human disease.

Authors:
Thomas A Milne

Blood 2017 04 8;129(16):2217-2223. Epub 2017 Feb 8.

Weatherall Institute of Molecular Medicine, Medical Research Council Molecular Haematology Unit, University of Oxford, Oxford, United Kingdom.

Chromosome translocations involving the mixed lineage leukemia () gene fuse it in frame with multiple partner genes creating novel fusion proteins (MLL-FPs) that cause aggressive acute leukemias in humans. Animal models of human disease are important for the exploration of underlying disease mechanisms as well as for testing novel therapeutic approaches. Patients carrying MLL-FPs have very few cooperating mutations, making MLL-FP driven leukemias ideal for animal modeling. The fact that the MLL-FP is the main driver mutation has allowed for a wide range of different experimental model systems designed to explore different aspects of MLL-FP leukemogenesis. In addition, MLL-FP driven acute myeloid leukemia (AML) in mice is often used as a general model for AML. This review provides an overview of different MLL-FP mouse model systems and discusses how well they have recapitulated aspects of the human disease as well as highlights the biological insights each model has provided into MLL-FP leukemogenesis. Many promising new drugs fail in the early stages of clinical trials. Lessons learned from past and present MLL-FP models may serve as a paradigm for designing more flexible and dynamic preclinical models for these as well as other acute leukemias.
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http://dx.doi.org/10.1182/blood-2016-10-691428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5399479PMC
April 2017

MLL-AF4 Spreading Identifies Binding Sites that Are Distinct from Super-Enhancers and that Govern Sensitivity to DOT1L Inhibition in Leukemia.

Cell Rep 2017 01;18(2):482-495

MRC, Molecular Haematology Unit, NIHR Oxford Biomedical Research Centre Programme, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK. Electronic address:

Understanding the underlying molecular mechanisms of defined cancers is crucial for effective personalized therapies. Translocations of the mixed-lineage leukemia (MLL) gene produce fusion proteins such as MLL-AF4 that disrupt epigenetic pathways and cause poor-prognosis leukemias. Here, we find that at a subset of gene targets, MLL-AF4 binding spreads into the gene body and is associated with the spreading of Menin binding, increased transcription, increased H3K79 methylation (H3K79me2/3), a disruption of normal H3K36me3 patterns, and unmethylated CpG regions in the gene body. Compared to other H3K79me2/3 marked genes, MLL-AF4 spreading gene expression is downregulated by inhibitors of the H3K79 methyltransferase DOT1L. This sensitivity mediates synergistic interactions with additional targeted drug treatments. Therefore, epigenetic spreading and enhanced susceptibility to epidrugs provides a potential marker for better understanding combination therapies in humans.
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http://dx.doi.org/10.1016/j.celrep.2016.12.054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5263239PMC
January 2017

MLL-AF4 binds directly to a BCL-2 specific enhancer and modulates H3K27 acetylation.

Exp Hematol 2017 03 14;47:64-75. Epub 2016 Nov 14.

Weatherall Institute of Molecular Medicine, MRC Molecular Haematology Unit, University of Oxford, Headington, Oxford, UK. Electronic address:

Survival rates for children and adults carrying mutations in the Mixed Lineage Leukemia (MLL) gene continue to have a very poor prognosis. The most common MLL mutation in acute lymphoblastic leukemia is the t(4;11)(q21;q23) chromosome translocation that fuses MLL in-frame with the AF4 gene producing MLL-AF4 and AF4-MLL fusion proteins. Previously, we found that MLL-AF4 binds to the BCL-2 gene and directly activates it through DOT1L recruitment and increased H3K79me2/3 levels. In the study described here, we performed a detailed analysis of MLL-AF4 regulation of the entire BCL-2 family. By measuring nascent RNA production in MLL-AF4 knockdowns, we found that of all the BCL-2 family genes, MLL-AF4 directly controls the active transcription of both BCL-2 and MCL-1 and also represses BIM via binding of the polycomb group repressor 1 (PRC1) complex component CBX8. We further analyzed MLL-AF4 activation of the BCL-2 gene using Capture-C and identified a BCL-2-specific enhancer, consisting of two clusters of H3K27Ac at the 3' end of the gene. Loss of MLL-AF4 activity results in a reduction of H3K79me3 levels in the gene body and H3K27Ac levels at the 3' BCL-2 enhancer, revealing a novel regulatory link between these two histone marks and MLL-AF4-mediated activation of BCL-2.
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http://dx.doi.org/10.1016/j.exphem.2016.11.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5333536PMC
March 2017

Instructive Role of MLL-Fusion Proteins Revealed by a Model of t(4;11) Pro-B Acute Lymphoblastic Leukemia.

Cancer Cell 2016 Nov;30(5):737-749

Department of Medicine, Section of Hematology/Oncology, University of Chicago, Chicago, IL 60637, USA. Electronic address:

The t(4;11)(q21;q23) fuses mixed-lineage leukemia (MLL) to AF4, the most common MLL-fusion partner. Here we show that MLL fused to murine Af4, highly conserved with human AF4, produces high-titer retrovirus permitting efficient transduction of human CD34 cells, thereby generating a model of t(4;11) pro-B acute lymphoblastic leukemia (ALL) that fully recapitulates the immunophenotypic and molecular aspects of the disease. MLL-Af4 induces a B ALL distinct from MLL-AF9 through differential genomic target binding of the fusion proteins leading to specific gene expression patterns. MLL-Af4 cells can assume a myeloid state under environmental pressure but retain lymphoid-lineage potential. Such incongruity was also observed in t(4;11) patients in whom leukemia evaded CD19-directed therapy by undergoing myeloid-lineage switch. Our model provides a valuable tool to unravel the pathogenesis of MLL-AF4 leukemogenesis.
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http://dx.doi.org/10.1016/j.ccell.2016.10.008DOI Listing
November 2016

MLL-AF9 Expression in Hematopoietic Stem Cells Drives a Highly Invasive AML Expressing EMT-Related Genes Linked to Poor Outcome.

Cancer Cell 2016 07 23;30(1):43-58. Epub 2016 Jun 23.

Department of Biomedicine, University Children's Hospital (UKBB), University of Basel, 4031 Basel, Switzerland. Electronic address:

To address the impact of cellular origin on acute myeloid leukemia (AML), we generated an inducible transgenic mouse model for MLL-AF9-driven leukemia. MLL-AF9 expression in long-term hematopoietic stem cells (LT-HSC) in vitro resulted in dispersed clonogenic growth and expression of genes involved in migration and invasion. In vivo, 20% LT-HSC-derived AML were particularly aggressive with extensive tissue infiltration, chemoresistance, and expressed genes related to epithelial-mesenchymal transition (EMT) in solid cancers. Knockdown of the EMT regulator ZEB1 significantly reduced leukemic blast invasion. By classifying mouse and human leukemias according to Evi1/EVI1 and Erg/ERG expression, reflecting aggressiveness and cell of origin, and performing comparative transcriptomics, we identified several EMT-related genes that were significantly associated with poor overall survival of AML patients.
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http://dx.doi.org/10.1016/j.ccell.2016.05.011DOI Listing
July 2016

MLL-Rearranged Acute Lymphoblastic Leukemias Activate BCL-2 through H3K79 Methylation and Are Sensitive to the BCL-2-Specific Antagonist ABT-199.

Cell Rep 2015 Dec 17;13(12):2715-27. Epub 2015 Dec 17.

Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

Targeted therapies designed to exploit specific molecular pathways in aggressive cancers are an exciting area of current research. Mixed Lineage Leukemia (MLL) mutations such as the t(4;11) translocation cause aggressive leukemias that are refractory to conventional treatment. The t(4;11) translocation produces an MLL/AF4 fusion protein that activates key target genes through both epigenetic and transcriptional elongation mechanisms. In this study, we show that t(4;11) patient cells express high levels of BCL-2 and are highly sensitive to treatment with the BCL-2-specific BH3 mimetic ABT-199. We demonstrate that MLL/AF4 specifically upregulates the BCL-2 gene but not other BCL-2 family members via DOT1L-mediated H3K79me2/3. We use this information to show that a t(4;11) cell line is sensitive to a combination of ABT-199 and DOT1L inhibitors. In addition, ABT-199 synergizes with standard induction-type therapy in a xenotransplant model, advocating for the introduction of ABT-199 into therapeutic regimens for MLL-rearranged leukemias.
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http://dx.doi.org/10.1016/j.celrep.2015.12.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4700051PMC
December 2015

Dangerous liaisons: cooperation between Pbx3, Meis1 and Hoxa9 in leukemia.

Haematologica 2015 Jul;100(7):850-3

MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, UK

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http://dx.doi.org/10.3324/haematol.2015.129932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4486217PMC
July 2015

Self-enforcing feedback activation between BCL6 and pre-B cell receptor signaling defines a distinct subtype of acute lymphoblastic leukemia.

Cancer Cell 2015 Mar;27(3):409-25

Division of Pediatric Hematology and Oncology, Department of Pediatrics, Oregon Health & Science University, Portland, OR 97239, USA; Papé Family Pediatric Research Institute, Oregon Health & Science University, Portland, OR 97239, USA.

Studying 830 pre-B ALL cases from four clinical trials, we found that human ALL can be divided into two fundamentally distinct subtypes based on pre-BCR function. While absent in the majority of ALL cases, tonic pre-BCR signaling was found in 112 cases (13.5%). In these cases, tonic pre-BCR signaling induced activation of BCL6, which in turn increased pre-BCR signaling output at the transcriptional level. Interestingly, inhibition of pre-BCR-related tyrosine kinases reduced constitutive BCL6 expression and selectively killed patient-derived pre-BCR(+) ALL cells. These findings identify a genetically and phenotypically distinct subset of human ALL that critically depends on tonic pre-BCR signaling. In vivo treatment studies suggested that pre-BCR tyrosine kinase inhibitors are useful for the treatment of patients with pre-BCR(+) ALL.
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http://dx.doi.org/10.1016/j.ccell.2015.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4618684PMC
March 2015

MLL5 expression as a biomarker for DNA hypermethylation and sensitivity to epigenetic therapy.

Authors:
Thomas A Milne

Haematologica 2014 Sep;99(9):1405-7

MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, UK

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http://dx.doi.org/10.3324/haematol.2014.113357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4562527PMC
September 2014
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