Characterising and targeting aberrant enhancer function in acute myeloid leukaemia (AML)

Lead Research Organisation: University of Cambridge
Department Name: Cambridge Institute for Medical Research

Abstract

Acute myeloid leukaemia (AML) is an aggressive haematological cancer. It is the most common acute leukaemia in Europe, with an incidence of 3-4 per 100,000 of the population. In addition, its incidence rises with age, therefore it will become an increasingly common problem as the general population ages. AML has a dismal overall survival rate of less than 30% and the mainstay of treatment aggressive, toxic, combination chemotherapy has remained ostensibly unchanged for the last 25 years. Novel therapies are therefore urgently required to improve treatment outcomes in AML. Where a greater knowledge of the mechanisms of disease have been uncovered, therapeutic gains have often been achieved. The most impressive example of this is the acute promyelocytic leukaemia subtype, where an understanding of the mechanisms that underlie this subtype has led to the development of novel therapies (All Trans Retionoic Acid, ATRA and arsenic trioxide) that can cure up to 90% of patients. Therefore, a better understanding of AML biology is a pre-requisite for the development of novel therapies to improve treatment outcomes.

Recently, a number of studies have documented that AML is associated with abnormal expression of genes and we and others have shown that correction of these abnormal gene programmes, with drugs such as small molecule inhibitors, may be a promising therapy. The expression of genes varies greatly between different tissues in the body and these differences are regulated by DNA elements called enhancers. Enhancers regulate the expression of genes by communication with promoters (the DNA start sites of genes) in a tissue-specific manner, such that for a gene X, there may be a number of different enhancers that regulate the expression of X in different tissues such as blood, brain and skin. Enhancers are a often a long distance away from promoters and require 3-dimensional contact with promoters through the formation of DNA loops. The function of enhancers is in turn controlled and modelled by molecules called transcription factors and regulators of chromatin (DNA and its protein scaffold). Recent work has shown that enhancers, transcription factors (TF) and chromatin regulators are all recurrently and commonly mutated in AML. We therefore propose that abnormal enhancer function is causative in AML and that it may be possible to therapeutically target this abnormal function to correct abnormal gene expression programmes thereby switching off leukaemia.

This proposal will address how enhancer usage and function differ during the evolution of AML from normal haematopoiesis, the process of normal blood formation. To allow us to model this prospectively, we will use mouse models with different combinations of AML-specific mutations that mimic different stages of the disease: normal, pre-leukaemic and frankly leukaemia stages. We will compare the presence, usage and function of enhancers between stages using the binding of specific proteins to DNA, a technique called ChIP-Seq. In addition, using another cutting-edge technique called Hi-C, we will map looping interactions between enhancers and promoters and how these differ by stage of disease. We will also measure the end output, that is expression of genes, detailing differences between the stages and how these correlate with enhancer usage. We will then compare these patterns to human leukaemia's that carry the same mutations. These data will allow us to identify the TF and chromatin regulators that control leukaemia specific enhancers. Our last objective will be to inhibit these factors, as a proof of concept that enhancer function may be a therapeutic target. We will use drug-like small molecular inhibitors and genetic techniques to alter the function of these regulators and will look for alterations of leukaemia growth and gene expression both in test tube experiments and live models of leukaemia.

Technical Summary

Acute Myeloid Leukaemia (AML) is an aggressive and often incurable haematological malignancy. Gene expression and mutational studies highlight the importance of abnormal transcription in the induction and maintenance of the leukaemia phenotype. Recent studies of normal transcription have documented the importance of specific enhancers, cis-regulatory elements often at long distances from the genes they regulate, in the induction and maintenance of tissue specific gene expression patterns. Growing evidence links aberrant enhancer function with malignancies such as AML and we propose to document alterations in the enhancer landscape that accompany the evolution of AML.

We will use an allelic series of sophisticated mouse strains that model the development of AML at the cellular level. Using pertinent haematopoietic stem and progenitor cell populations from each of these models, we will document alterations in gene expression, enhancer usage and function and enhancer-promoter interactions using state-of-the-art global genomics techniques such as RNA-Seq, ChIP-Seq and Capture Hi-C (CHI-C). We will then correlate the changes in our murine models with the enhancer landscape of human AML with a similar genotype. Finally, we will aim to identify transcription factors and chromatin rmodifiers that are critical for AML enhancer function and will test the therapeutic potential of perturbing these critical regulators with experimental and pharmacological intervention.

Planned Impact

Although Acute Myeloid Leukaemia (AML) is a relatively rare disease, it is highly aggressive. Of the 2200 or so patients diagnosed each year in the UK, around 1800 will not survive long-term. AML is therefore an unmet medical need. In addition to this social burden to the UK and beyond, it places a significant financial burden on the NHS. Curative treatment is highly intensive, which is arduous for the patient, expensive (over £60K/patient) and requires prolonged hospital admissions, representing a significant cost to the NHS. Allogeneic stem cell transplantation (alloSCT) provides a powerful approach to reduce relapse, but again is costly (£50-100K/ transplant) and is associated with significant toxicity (an upfront mortality rate of between 10-30%). The mainstay of treatment, standard combination chemotherapy, appears to have reached the ceiling of its impact, therefore, there is an urgent need for novel therapeutics in AML. However, where the biology of the disease is well characterized, such as in the acute promyelocytic leukaemia (APML) subtype, improvements have been dramatic, with cure rates now exceeding 90% for this subtype. Theses data prioritise a better understanding of AML biology as a pre-requisite for the development of novel therapies to improve treatment outcomes.

Recently, it has been demonstrated that the generation of tissue specific patterns of gene expression, which occurs during development and differentiation, involves the interplay of combinatorial patterns of specific enhancer elements with their cognate promoters. We predict that the normal remodelling of enhancers, required to generate specialised transcriptional programmes necessary for haematopoietic differentiation and development, is corrupted during the evolution of leukaemia, giving rise to aberrant transcription which maintains the tumour. In the majority of AML cases, although we know that transcription is altered to drive the leukaemic phenotype, the mechanisms that initiate a maintain these aberrant transcriptional programmes are poorly understood. Details of transcriptional alterations during leukaemia evolution, the mechanisms involved and how these might be therapeutically targeted, would not only inform leukaemogenesis and the normal transcriptional regulation of haematopoiesis, but may also identify novel therapeutic targets and therapies themselves.

Publications


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Basheer F (2015) BET bromodomain inhibitors in leukemia. in Experimental hematology
Gallipoli P (2015) Epigenetic regulators as promising therapeutic targets in acute myeloid leukemia. in Therapeutic advances in hematology
Gilan O (2016) Functional interdependence of BRD4 and DOT1L in MLL leukemia. in Nature structural & molecular biology
 
Description Academic representative on national JRCPTB Haematology specialist advisory committee (SAC)
Geographic Reach Local/Municipal/Regional - UK Only 
Policy Influence Type Influenced training of practitioners or researchers
 
Description NCRI AML working group
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
 
Description NCRI Chronic Myeloid Leukaemia (CML) working party
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
Impact To define and implement treatment policy for the UK clinical community in CML. To decide upon the nature of national trials in this disease
 
Description ERC Consolidator award
Amount € 2,000,000 (EUR)
Funding ID 647685 
Organisation European Research Council (ERC) 
Sector Public
Country European Union (EU)
Start 09/2015 
End 08/2020
 
Title Blast crisis progression model 
Description This is a novel mouse model that allows identification of mechanisms of blast crisis progression, using transposon-based insertional mutagenesis on the background of chronic phase Chronic Myeloid Lymphoma as published in Giotopoulos et al. J Exp Med 2015. 
Type Of Material Computer model/algorithm 
Year Produced 2015 
Provided To Others? Yes  
Impact The model greatly informs the biology of Chronic Myeloid Lymphoma progression and provides a potent resource for the development of candidate therapies to improve the dismal outcomes in this highly aggressive disease. 
 
Description Application of CREBBP/p300 inhibitor programme to haematological malignancies 
Organisation CellCentric Ltd
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Private 
PI Contribution Expertise in researching the biology of CREBBP/Crebbp as it relates to HSC function and its subversion in haematological malignancies. Relevant cell lines and mouse models.
Collaborator Contribution Making available a CREBBP/p300 inhibitor which they have developed
Impact It is too early for any concrete outputs.
Start Year 2017
 
Description Study of MLL-AF4+ infant B cell acute lymphoblastic leukaemia 
Organisation Medical Research Council (MRC)
Department MRC Centre for Regenerative Medicine
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Public 
PI Contribution Assistance with experiments, particularly assays and mouse modelling, and also data analysis.
Collaborator Contribution The majority of experiments and overall direction of the study.
Impact Study published as Barrett et al. Cell Rep 2016 increases our knowledge of a previously poorly understood childhood cancer and identifies of how future study could increase this further still.
Start Year 2015
 
Description Study of the leukemogenetic potential of novel and rare gene fusions 
Organisation University of Bologna (Università di Bologna UNIBO)
Country Italy, Italian Republic 
Sector Academic/University 
PI Contribution Expertise in mouse modelling and advice with experimental design.
Collaborator Contribution Production of gene fusions e.g. ZEB2-BCL11B
Impact The study should improve our understanding of novel and rare gene fusions and explore their potential as innovative targets for personalized therapies approaches within Acute Myeloid Leukaemia.
Start Year 2017
 
Description Using chromosome conformation capture analysis to study fumarate 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Public 
PI Contribution Performing chromosome conformation capture experiments
Collaborator Contribution Wider study of the role of fumarate in the development of renal cancer
Impact The study has been published (Sciacovelli et al. Nature 2016) showing loss of fumarate hydratase and fumarate accumulation contribute to the aggressive features of fumarate hydratase-deficient tumours with important implications for their future treatment.
Start Year 2015
 
Description Cambridge Science festival 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Structured talk and open discussion thereafter

Sparked lab visits and a video re blood cancers which will be filmed by our stem cell institute.
Year(s) Of Engagement Activity 2015