Is Bcr-Abl expression relevant for the survival of cancer stem cells in chronic myeloid leukaemia

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary &Life Sci

Abstract

CML has an incidence of 1.3 per 100,000 of the population with 750 new cases per year in the UK. CML begins when a stem or founder bone marrow cell develops a genetic change during division. This change results in the Philadelphia chromosome and the Bcr-Abl cancer protein being found in all CML cells, including the stem cells. The Bcr-Abl protein is an enzyme called a tyrosine kinase and tyrosine kinase inhibitors (TKIs) have now been introduced for the treatment of CML. Imatinib mesylate (IM), the first of these drugs to reach the clinic, is now the standard of care for 85% of CML patients in the early stages of the condition. When started at diagnosis this drug has induced remission in 80% of cases, leading to absence of the Ph chromosome, and has reduced the annual rate of progression from 10-15%, before the introduction of IM, to only 2-4%, with patients now expected to survive at least 10 years from diagnosis and possibly much longer. However IM does not cure CML and all patients still have CML stem cells present. We have recently shown that the amount of Bcr-Abl cancer protein is greatly increased in CML stem cells and we were the first to show that CML stem cells are resistant to killing by IM. Our current work is investigating whether IM, or new generation stronger TKIs, achieve high enough levels inside the CML stem cells to effectively overcome the very high levels of cancer protein and whether drug pumps, on the surface of CML stem cells, limit the build up of these drugs inside cells. The question we wish to address in this proposal is whether the activity (whether it is on or off) of the Bcr-Abl cancer protein is the major determinant of whether these stem cells survive and divide to sustain leukaemia in patients or undergo a process called programmed cell death when the protein is switched off. We are confident we have the expertise to investigate and answer this question. If CML stem cell survival does not require Bcr-Abl to be on at all times then we need to develop new approaches, other than TKIs, to kill these cells. Our overall aim is to understand what keeps CML stem cells alive and find new ways to kill these cells so that patients may be cured and come off all drug therapies.

Technical Summary

Chronic myeloid leukaemia (CML) arises in a haemopoietic stem cell targeted by the t(9;22)/Ph translocation. Although all Ph+ cells express Bcr-Abl transcripts and oncoprotein, we have recently shown both mRNA and oncoprotein to be greatly overexpressed at the stem cell level. Bcr-Abl is a constitutively active tyrosine kinase and a number of tyrosine kinase inhibitors (TKIs) have now been introduced for the treatment of CML. We were the first to show that CML stem cells are inherently insensitive to imatinib mesylate (IM), the first of these agents to reach the clinic. Furthermore, IM does not eradicate CML stem cells in vivo and the majority of patients have residual disease as detected by RT-PCR for Bcr-Abl transcripts. Our current work has investigated whether IM, or more potent TKIs, achieve high enough intracellular levels within CML stem cells to effectively inhibit the upregulated levels of oncoprotein and the role that influx and efflux drug pumps play in determining intracellular drug accumulation. The key question that will be addressed here is whether Bcr-Abl activity is relevant for survival of CML stem cells. One hypothesis is that by inhibiting Bcr-Abl in a stem cell you simply revert the malignant phenotype to normal. The alternative hypothesis is that at the time the stem cell acquires t(9;22), additional changes occur that confer a survival advantage and the cell becomes dependent on Bcr-Abl expression. We are now confident we have the expertise to investigate and answer this question. We will use alternative but complementary approaches. Primary CD34+ CML cells will be exposed to TKIs in vitro with surviving stem cells tracked over time, at the single cell level, for inhibition of Bcr-Abl activity, apoptosis, retention of primitive phenotype and function and proliferation. To maximise Bcr-Abl inhibition at the stem cell level the above approach will be combined with lentiviral delivered shRNA against Bcr-Abl. Finally to demonstrate the effect of Bcr-Abl inhibition within transplantable stem cells in vivo, a transgenic model that expresses Bcr-Abl from a tetracycline responsive element driven by the SCL3? enhancer (to allow induced expression of Bcr-Abl in the stem cell compartment), will be employed. If CML stem cell survival is proven to be independent of Bcr-Abl, then alternative approaches to selectively target and eradicate the cancer stem cell pool will be required. For example, these might exploit Bcr-Abl expression to facilitate delivery of a cytotoxic agent selectively to Ph+ CML cells.

Publications

10 25 50
 
Description training of basic pHd and clinical research fellows
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Influenced training of practitioners or researchers
 
Description CRUK
Amount £19,000 (GBP)
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2012 
End 02/2013
 
Description KKLF
Amount £330,216 (GBP)
Organisation The Kay Kendall Leukaemia Fund 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2010 
End 09/2013
 
Description MRC Clinical Trial Grant (A randomised phase 2 trial of IM versus HCQ/IM with CML in MCyR with residual disease by Q-PCR)
Amount £833,326 (GBP)
Funding ID G0900882 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 09/2010 
End 09/2013
 
Description MRC DTG
Amount £19,980 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 09/2014 
End 02/2015
 
Description Novartis
Amount £60,000 (GBP)
Organisation Novartis 
Sector Private
Country Global
Start 01/2011 
End 01/2013
 
Description Programme grant
Amount £1,700,000 (GBP)
Organisation Bloodwise 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2015 
End 05/2020
 
Description SULSA
Amount £100,000 (GBP)
Funding ID MSD23_G_Holyoake-Chan 
Organisation Scottish Universities Life Sciences Alliance 
Sector Academic/University
Country United Kingdom
Start 10/2012 
End 09/2016
 
Description Bruno Calabretta 
Organisation Thomas Jefferson University
Department Department of Cancer Biology
Country United States 
Sector Academic/University 
PI Contribution published JCI paper together
Collaborator Contribution ENHANCED RESEARCH
Impact JCi paper Kay Kendall grant MRC grant MRC DGT sponsorship
Start Year 2007
 
Description CRUK 
Organisation Beatson Institute for Cancer Research
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution We are the leaders of partnership
Collaborator Contribution Providing LC3-GFP mouse to cross to BCR-ABL double transgenic mouse. Ed Chan is providing expertise relating to ULK-1
Impact Nothing as yet
Start Year 2012
 
Description Paolo Salmoni 
Organisation Medical Research Council (MRC)
Department MRC Toxicology Unit
Country United Kingdom 
Sector Public 
PI Contribution we published the JCI paper together
Collaborator Contribution enhanced science
Impact JCI paper Kay kendall grant MRC grant MRC DGT
Start Year 2007
 
Description SULSA 
Organisation Scottish Universities Life Sciences Alliance
Country United Kingdom 
Sector Academic/University 
PI Contribution We are the leaders of the partnership
Collaborator Contribution Ed Chan is providing expertise relating to ULK-1
Impact Still awaitng
Start Year 2012
 
Description autophagy work 
Organisation Thomas Jefferson University
Country United States 
Sector Academic/University 
PI Contribution We have generated a new grant entitled "A randomised phase 2 trial of IM versus HCQ/IM for patients with CML in MCyR with residual disease by Q-PCR" MRC Ref: G0900882
Collaborator Contribution Collaborated on a paper - Pub med reference 19363292
Impact 19363292
Start Year 2009
 
Description autophagy work 
Organisation University College London
Department UCL Cancer Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution We have generated a new grant entitled "A randomised phase 2 trial of IM versus HCQ/IM for patients with CML in MCyR with residual disease by Q-PCR" MRC Ref: G0900882
Collaborator Contribution Collaborated on paper - Pubmed reference 19363292
Impact 19363292
Start Year 2009
 
Description 4th International Workshop on CML, Natchez 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact Talk

Improved collaborations
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Description AAAS Vancouver 
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Description ASH San Diego 
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Description Australia Directors in Leukaemia Meeting 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Health professionals
Results and Impact audience 200, mix of scientists and medics,45 minutes

improved collaborations
Year(s) Of Engagement Activity 2008
 
Description BUSCC University Stem Cell seminars birmingham 
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Description BUSCC University Stem cell seminar Birmingham 
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Description Beatson Institute for Cancer Research Glasgow 
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Description Beatson Institute for Cancer Research, Glasgow 
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Description Beatson Institute, Cancer School, Glasgow 
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Description British Society of Haematology 
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Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach National
Primary Audience Participants in your research and patient groups
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Improved collaborations
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Description British Society of Haematology - Brighton 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Health professionals
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Improved collaborations
Year(s) Of Engagement Activity 2009
 
Description CML Professionals Meeting Newcastle 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach National
Primary Audience Participants in your research and patient groups
Results and Impact CML Professionals Meeting

Improved collaborations
Year(s) Of Engagement Activity 2012
 
Description Cambridge 
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Year(s) Of Engagement Activity 2009
 
Description Cancer Research Award Symposium, Germany 
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Geographic Reach International
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Year(s) Of Engagement Activity 2009
 
Description EHA Amsterdam 
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Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact Talk

Improved collaborations
Year(s) Of Engagement Activity 2012
 
Description EHA London 
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Results and Impact Talk

Improved collaborations
Year(s) Of Engagement Activity 2011
 
Description EHA Milan 
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Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact talk with discussion

improved collaborations
Year(s) Of Engagement Activity 2014
 
Description EHA meeting London 
Form Of Engagement Activity A talk or presentation
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Geographic Reach International
Primary Audience Participants in your research and patient groups
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Year(s) Of Engagement Activity 2011
 
Description EHA/ESH Cancer Stem cell workshop, Cannes, France 
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Part Of Official Scheme? No
Geographic Reach International
Primary Audience Health professionals
Results and Impact Talk

Improved collaborations
Year(s) Of Engagement Activity 2009
 
Description ESH 
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Results and Impact 500 audience, CML research and clinical run by ESH

improved collaborations
Year(s) Of Engagement Activity 2008
 
Description ESH Baltimore 
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Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact Talk

Improved collaborations
Year(s) Of Engagement Activity 2012
 
Description ESH Portugal 
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Year(s) Of Engagement Activity 2011
 
Description ESH Vienna 
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Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact Talk

Improved collaborations
Year(s) Of Engagement Activity 2012
 
Description ESH-CML Bordeaux 
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Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact Talk

Improved collaborations
Year(s) Of Engagement Activity 2009
 
Description Erasmus Haematology Lecture 
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Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Participants in your research and patient groups
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Improved collaborations
Year(s) Of Engagement Activity 2013
 
Description French Haematology group 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Health professionals
Results and Impact audience of 120, French haematologists and clinical trial co-ordinators, 30 minutes

improved collaborations
Year(s) Of Engagement Activity 2008
 
Description ISEH Amsterdam 
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Part Of Official Scheme? No
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Geographic Reach International
Primary Audience Participants in your research and patient groups
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Year(s) Of Engagement Activity 2012
 
Description LR, GHD London 
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Year(s) Of Engagement Activity 2009
 
Description LRF one off meeting 
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Primary Audience Health professionals
Results and Impact around 200, scientist and medical staff, 25 minutes,

press release
Year(s) Of Engagement Activity 2008
 
Description Leukaemia and cancer stem cells workshop 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
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Results and Impact small workshop only 23, biologists, computer modellers, mathematicians

improved collaborations
Year(s) Of Engagement Activity 2008
 
Description NCMLSG Seminar Stockholm 
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Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact talk

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Year(s) Of Engagement Activity 2011
 
Description NCMLSG Stockholm 
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Primary Audience Participants in your research and patient groups
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Year(s) Of Engagement Activity 2011
 
Description NCRI annual meeting Birmingham 
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Primary Audience Health professionals
Results and Impact 2-300, oncologists, haematologists, public, patients, nurses, pharmacists etc

did a report for press
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Description Open Day 
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Geographic Reach Local
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Results and Impact audience of 25, short speeches and lab demonstrations

press release
Year(s) Of Engagement Activity 2008
 
Description Paterson Institute Manchester 
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improved collaborations
Year(s) Of Engagement Activity 2011
 
Description Paterson Institute Manchester 
Form Of Engagement Activity A talk or presentation
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Year(s) Of Engagement Activity 2011
 
Description Quarterley Divisional newsletter 
Form Of Engagement Activity A magazine, newsletter or online publication
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Results and Impact Update with all grants and papers

Information circulated to all scientists within division
Year(s) Of Engagement Activity 2009
 
Description South East Scotland Blood Club meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Health professionals
Results and Impact talk

opened up discussion
Year(s) Of Engagement Activity 2014
 
Description TSCRC Dundee 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Participants in your research and patient groups
Results and Impact Talk

Improved collaborations
Year(s) Of Engagement Activity 2011
 
Description Third Global Workshop on CML, New Orleans 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Health professionals
Results and Impact Talk

Improved collaborations
Year(s) Of Engagement Activity 2009
 
Description UCSF Cancer Centre, San Francisco 
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Primary Audience Participants in your research and patient groups
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Year(s) Of Engagement Activity 2011
 
Description UCSF Cancer Centre, San Francisco 
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Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact Talk

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Year(s) Of Engagement Activity 2011
 
Description UKNSCN 4th Annual science meeting York 
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Year(s) Of Engagement Activity 2011
 
Description UKNSCN York 
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Primary Audience Participants in your research and patient groups
Results and Impact Talk

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Year(s) Of Engagement Activity 2011
 
Description Vienna Cancer Stem Cell Symposium 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Health professionals
Results and Impact 100, scientists and medical staff

improved collaborations
Year(s) Of Engagement Activity 2008
 
Description Wilsede cancer stem cells meeting 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Health professionals
Results and Impact audience 200, students, scientists, medical staff, interactive,

improved collaborations
Year(s) Of Engagement Activity 2008
 
Description lecture tour Canada 
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Part Of Official Scheme? No
Geographic Reach International
Primary Audience Health professionals
Results and Impact 9 lectures in Montreal, Ottawa, Toronto, Hamilton between 8-11 Sept 2008, medical staff, students, nurses

improved collaborations
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Description open days for new paul O'Gorman Leukaemia Research centre 
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Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact 9/10 and 22 May 2008. Audience of around 100 at each, short speeches, poster presentations, demon strations in the laboratory

press were present - Sunday Times, Herald articles guest of honour Richard Rockefeller
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Description retiral lecture 
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Primary Audience Health professionals
Results and Impact audience around 80, local cytogenetics department for retiral of member of staff

improved collaborations
Year(s) Of Engagement Activity 2008