Role of the Notch signalling pathway in acute myeloid leukaemia

Lead Research Organisation: University College London
Department Name: Unlisted

Abstract

We are trying to identify the role that a gene called Notch plays in acute leukaemia. We already know that over half of children with T-cell acute lymphoblastic leukaemia (T-ALL) have mutations in Notch-1, but their role in the causation of the disease is not completely understood.

The Notch pathway plays several roles in normal blood cell production, being particularly important in the production of T-cells and blood stem cells.

Leukaemic cells are unable to develop into normal blood cells. Our preliminary studies suggest that a gene called CSL, which binds to Notch, may prevent normal cell development. By inserting this gene into normal and leukaemic cells in culture, we hope to gain an understanding as to how, and why, this might be the case. We will also explore the possibility that other members of the Notch family are involved in leukaemia development.
We are particularly interested in addressing whether Notch-1 mutations occur in all age groups of patients with T-ALL and whether they are involved in other forms of acute leukaemia apart from T-ALL. This is particularly timely as drugs that inhibit Notch are in development for the treatment of T-ALL.
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Technical Summary

The Notch pathway, pivotal in directing cell fate in embryonic development, is now recognised as having an important role in regulation of haemopoiesis. Notch directs the transition from common lymphoid stem cell to committed T-cell progenitor, and has recently been shown to regulate the stem cell niche, affect myeloid differentiation, and control haemopoietic stem cell self-renewal, all of which are dysregulated in leukaemogenesis. It is a membrane-bound receptor; on ligand binding its intracellular portion is cleaved and translocates to the nucleus to bind the transcription factor CSL. It has recently been discovered that >50% of paediatric/adolescent patients with T-cell acute lymphoblastic leukaemia (T-ALL) have activating mutations in the Notch-1 receptor in either the heterodimerisation or PEST domain, and sometimes in both domains. Notch-3, which shares many features with Notch-1, including its ability to produce a T-cell leukaemia on transfection into mice, is a likely target gene in those cases of T-ALL without a Notch-1 mutation. We hypothesise that mutations of the Notch pathway may be found in acute myeloid leukaemia (AML) patients where there is aberrant T-cell expression. Infants with leukaemia often have mixed lineage surface antigen expression and this rare group of patients also warrants detailed analysis. Additionally, we have found that ˜50% of patients with AML have a previously undescribed isoform of CSL where exon 10 is truncated (termed CSL-TREX). When AML blasts can be induced to differentiate in vitro, CSL-TREX is down-regulated, thus raising the possibility that it contributes to maintenance of the undifferentiated state in vivo.

Samples from a cohort of infant leukaemia patients, accessed from the UCL Institute of Child Health DNA bank, will be screened for Notch-1 and Notch-3 mutations by heteroduplex analysis, and positive samples sequenced. Notch-1 will be studied in 50 adult AML patients with aberrant T-cell expression and comparedwith 50 patients without these markers, as identified from the MRC DNA bank. In order to explore the possibility of autocrine stimulation, the Notch ligand Jagged-1, Notch-1 and the downstream molecule HES-1, will be quantified by real-time RT-PCR of 200 AML RNA samples. We will use a single cell, Notch-sensitive, GFP-reporter system to analyse Notch activation in de novo AML cells. Finally, we will further characterise the function of CSL-TREX by transfecting CD34+ cells using a GFP-expressing lentiviral vector. These studies are particularly timely as -secretase inhibitors that inhibit Notch function are already in phase I/II trials in childhood T-ALL.

Publications

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Li N (2014) Cyclin C is a haploinsufficient tumour suppressor. in Nature cell biology

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Mansour MR (2009) Prognostic implications of NOTCH1 and FBXW7 mutations in adults with T-cell acute lymphoblastic leukemia treated on the MRC UKALLXII/ECOG E2993 protocol. in Journal of clinical oncology : official journal of the American Society of Clinical Oncology

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Mansour MR (2007) Notch-1 mutations are secondary events in some patients with T-cell acute lymphoblastic leukemia. in Clinical cancer research : an official journal of the American Association for Cancer Research

 
Description Citation in American Society of Hematology Educational Review on T-ALL
Geographic Reach Multiple continents/international 
Policy Influence Type Citation in clinical reviews
 
Description KKLF Intermediate Fellowship
Amount £570,000 (GBP)
Organisation The Kay Kendall Leukaemia Fund 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2010 
End 09/2013
 
Title T-ALL sample bank 
Description DNA samples were never collected prospectively on the UK MRC UKALLXII trial, despite patients consenting to collection and molecular analysis at the time of trial entry. By contacting sites around the country, I collected 54 diagnostic DNA samples from patients with T-ALL, all uniformly treated on the trial. Correlation of molecular abnormalities with prognostic outcome and clinical variables in this rare disease is now possible. 
Type Of Material Database/Collection of Data/Biological Samples 
Year Produced 2008 
Provided To Others? Yes  
Impact Together with the 34 patients collected by our collaborators from ECOG (joint trial), we have one of the largest collection of adult T-ALL samples treated on the same trial. This has led to two notable publications : PMID: 19494353 and PMID: 19635999. 
URL http://europepmc.org/abstract/MED/19494353
 
Description Adolfo Ferrando group 
Organisation Columbia University
Country United States 
Sector Academic/University 
PI Contribution DNA samples from the ECOG2993/MRC UKALLXII trial for acute lymphoblastic leukaemia were never prospectively collected or stored. I collected 54 samples of UK treated ALL patients from the trial and analysed Notch and FBXW7 mutation status. We collaborated with Adolfo Ferrando's group from Columbia University, New York, who had data on 34 patients treated on the same trial enabling us to combine the data for meaningful statistical analysis of prognostic outcome.
Collaborator Contribution Added data on 34 patients to our 54 patients treated on the same trial.
Impact This resulted in two publications: PubMedID 19494353 and 19635999
Start Year 2007
 
Description CSL 
Organisation Brigham and Women's Hospital
Department Department of Pathology
Country United States 
Sector Hospitals 
PI Contribution We identified a novel isoform of CSL which Jon Aster's group is helping us characterise
Collaborator Contribution Performing biochemical analysis of the CSL interacting partners
Impact Now have enough data from our combined studies to submit for publication. Aiming for submission to JEM.
Start Year 2007
 
Description CSL isoform characterisation 
Organisation Dana-Farber Cancer Institute
Department Department of Pediatric Oncology
Country United States 
Sector Academic/University 
PI Contribution Identified a novel isoform of CSL
Collaborator Contribution Novel isoform of CSL has been tested in vivo at DFCI
Impact Now have adequate data to publish. Plan to submit to JEM
Start Year 2010
 
Description Stephen Blacklow Group 
Organisation Brigham and Women's Hospital
Country United States 
Sector Hospitals 
PI Contribution We identified an activating mutation in the LNR domain, the first ever to be reported in this domain of Notch-1, in a patient with T-ALL. Blacklow and Aster's group solved the crystal structure of Notch-1 and by mapping our novel mutation, were able to make important predictions on functionality of the LNR domain in terms of Notch activation.
Collaborator Contribution Blacklow's group solved the crystal structure of Notch-1 enabling our novel mutation to be structurally and functionally analysed.
Impact Publication Pubmed ID 19075186
Start Year 2007
 
Description CRUK Fundraisers 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Primary Audience Public/other audiences
Results and Impact Fundraisers (Dallaglio Foundation) for CRUK were interested in what was involved in a Clinical Research Training Fellowship and the research outcome from my work.

The Dellaglio Foundation managed to raise £650,000 specifically for Clinical Research Training Fellowships for CRUK
Year(s) Of Engagement Activity 2009