Role of reactive oxygen species in modulating carbohydrate metabolism in acute myeloid leukaemia

290 Pages·2017·4.75 MB·English

by  Andy Robinson

Role of reactive oxygen species in
modulating carbohydrate
metabolism in acute myeloid
leukaemia

by
Andrew J Robinson

2017

A thesis presented to Cardiff University in partial
fulfilment of the requirement for the degree of
Doctor of Philosophy

Department of Haematology,
School of Medicine,
Cardiff University.

Supervisors: Dr. Alex Tonks and Professor R Darley
Declaration and statements
DECLARATION
This work has not been submitted in substance for any other degree or award at this
or any other university or place of learning, nor is being submitted concurrently in
candidature for any degree or other award.
Signed............................... (candidate) Date.............................

STATEMENT 1
This thesis is being submitted in partial fulfilment of the requirements for the degree
of Doctor of Philosophy (PhD).
Signed............................... (candidate) Date.............................

STATEMENT 2
This thesis is the result of my own independent work/investigation, except where
otherwise stated, and the thesis has not been edited by a third party beyond what is
permitted by Cardiff University’s Policy on the Use of Third Party Editors by
Research Degree Students. Other sources are acknowledged by explicit references.
The views expressed are my own.
Signed............................... (candidate) Date.............................

STATEMENT 3
I hereby give consent for my thesis, if accepted, to be available online in the
University’s Open Access repository and for inter-library loan, and for the title and
summary to be made available to outside organisations.
Signed............................... (candidate) Date.............................

STATEMENT 4: PREVIOUSLY APPROVED BAR ON ACCESS
I hereby give consent for my thesis, if accepted, to be available online in the
University’s Open Access repository and for inter-library loans after expiry of a bar
on access previously approved by the Academic Standards & Quality Committee.
Signed............................... (candidate) Date.............................
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Acknowledgements

I would like to express my sincerest thanks to my supervisors Dr. Alex Tonks and Prof.
Richard Darley. Their knowledge, guidance and inexhaustible patience, alongside their
unswerving support and encouragement have been essential in completing this study.
I would also like to thank Dr’s Paul Hole, Nader Omidvar and Chinmay Munje, for their
generous assistance in providing knowledge around particular aspects of this project.
Grateful thanks also to Sara Davies and Sarah Baker for their technical assistance, for
‘showing me the laboratory ropes’ and for making me feel welcome.
I wish also to acknowledge the contribution of Prof. Awen Gallimore, who convinced
me I still had what it takes to engage in academic research.
In addition, a huge thank-you to my family and friends, for their laughter (both with and
at me), their endless encouragement and their belief in my ability.
Finally, it would be remiss of me not to expressly acknowledge the I.T. expertise
provided by Dr. Alex Tonks, without which this document may well have been
presented on Vellum.
Ultimately, none of this work would have been possible without the generosity of
Tenovus Cancer Care who funded this research, and in particular the volunteers who
tirelessly collect, in the pursuit of curing cancer. My thanks to them all.

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This work is dedicated to my wife Katie and my children, Seren and Carys.
I wish to thank them from the bottom of my heart.

Ever tried. Ever failed. No matter. Try again. Fail again. Fail better.
- Samuel Beckett

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Publications and presentations

Abstracts
Hopkins, G.L., Robinson A.J., Hole, P. S., Darley, R. L., & Tonks, A. Analysis of ROS
responsive genes in mutant RAS expressing hematopoietic progenitors identifies the
glycolytic pathway as a major target promoting both proliferation and survival.
American Society of Haematology Annual Meeting, December 2014. (Accompanied by
poster presentation).

Oral presentations
Robinson, A.J., Kreuser, S., Davies, S., Darley, R., & Tonks, A. Reactive oxygen
species increases glycolysis in acute myeloid leukaemia via overexpression of PFKFB3.
17th International Conference on Oxidative Stress Reduction, Redox Homeostasis and
Antioxidants, June 2016.

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Abstract
Reactive oxygen species (ROS) are known to play a significant role in cellular
signalling pathways, associated with growth, differentiation and survival and in many
cancers, elevated ROS levels have been linked with aberrant signalling. Previously, we
showed that human haematopoietic progenitor cells expressing mutant RAS (a common
abnormality in AML) had elevated NOX2 derived ROS which led to increased cell
proliferation. However, it is currently unknown how ROS mediates these effects.
Previous preliminary data suggests significant ROS attributable gene changes are
associated with glycolysis, a feature also common in solid tumours. Using human
haematopoietic progenitor cells, it is reported here for the first time that, elevated ROS
leads to changes in extracellular lactate production and increases in glucose uptake.
Furthermore, modulation of ROS levels in AML cell lines also generate results
consistent with a ROS induced model of increased glycolysis. Gene expression profiling
identified ROS related changes in specific glycolytic enzymes, whilst immunoblotting
verified ROS dependent increases in protein expression of a key regulatory glycolytic
enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), in both
human cells and AML cell lines. Metabolomic analysis using mass spectrometry, was
performed on AML cell lines in which ROS levels were manipulated and AML patient
samples were characterised according to ROS production. This revealed changes in
concentrations of metabolites associated with glycolysis and metabolic pathways
important in ROS regulation. Modulation of PFKFB3 expression generated changes in
glucose uptake consistent with ROS mediated changes in this enzyme. In summary, this
study establishes for the first time that increased ROS production in AML models leads
to increased glycolysis and metabolic reprogramming as a consequence of modulation
of PFKFB3 expression by ROS.

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List of Abbreviations
1,3-BPG 1,3-bisphosphoglycerate
2-NBDG 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-
D-deoxyglucose
2-PG 2-phosphoglcyerate
3-PG 3-phosphoglycerate
3PO 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one
6-PG 6-phosphogluconolactone
6-PGD 6-phosphogluconate dehydrogenase
7-AAD 7-aminoactinomycin D
ADP adenosine diphosphate
ALDO aldolase
ALL acute lymphocytic leukaemia
AKT protein kinase B
AML acute myeloid leukaemia
AMPK 5’ adenosine monophosphate-activated protein kinase
ARA-C Cytarabine
ATP adenosine triphosphate
BM bone marrow
BSA bovine serum albumin fraction V
CD cluster of differentiation
CLL chronic lymphocytic leukaemia
CML chronic myeloid leukaemia
DHAP dihydroxyacetone phosphate
DMEM Dulbecco Modified Eagle Medium
DPI diphenyleneiodonium
ENO enolase
ERK extracellular signal-regulated proteins
ETC electron transport chain
F-6-P fructose-6-phosphate
F-1,6-BP fructose-1,6-bisphosphate
F-2,6-BP fructose-2,6-bisphosphate
FACS fluorescence activated cell sorting
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FBS foetal bovine serum
G-3-P glyceraldehyde-3-phosphate
G-6-P glucose-6-phosphate
G-6-PD glucose-6-phosphate dehydrogenase
GAP GTPase activating proteins
GAPDH glyceraldehyde-3-phosphate dehydrogenase
G-CSF granulocyte-colony stimulating factor
GDP guanosine diphosphate
GEF guanine nucleotide exchange factor
GFP green fluorescent protein
GLUT glucose transporter
GM-CSF granulocyte macrophage-colony stimulating factor
GOX glucose oxidase
GPX glutathione peroxidase
GSH glutathione
GSR glutathione reductase
GSSG glutathione disulphide
GTP guanosine triphosphate
GTPase guanosine triphosphatase
H O hydrogen peroxide
2 2
HIF-1α hypoxia inducible factor-1 alpha
HK hexokinase
HOCl hypochlorous acid
HPC haematopoietic progenitor cells
HSC haematopoietic stem cells
IL-3 interleukin-3
IL-6 interleukin-6
IMDM Iscove’s Modified Dulbecco’s Medium
LDH lactate dehydrogenase
LSC leukaemia stem cells
MAPK mitogen activated protein kinase
MCT monocarboxylate transporter
MEK meiosis specific serine/threonine-protein kinase
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mTOR mammalian target of rapamycin
NADPH nicotinamide adenine dinucleotide phosphate
NF-κB nuclear factor kappa-light-chain-enhancer of
activated B-cells
NOX NADPH oxidase
Nrf2 nuclear-related factor 2
O diatomic oxygen
2
O •- superoxide
2
OH• hydroxyl radical
PBS phosphate buffered saline
PEP phosphoenolpyruvate
PFK phosphofructokinase
PFK158 1-(pyridine-4-yl)-3-(7-(trifluoromethyl)-quinolin-2-
yl)-prop-2-en-1-one
PFKFB3 6-phosphofructo-2-kinase/fructose-2,6-
bisphosphatase 3
PGAM phosphoglycerate mutase
PGI phosphoglucose isomerase
PGK phosphoglycerate kinase
PHGDH phosphoglycerate dehydrogenase
PI3K phosphatidylinositol 3-kinase
PKM2 pruvate kinase muscle 2
PPP pentose phosphate pathway
Prx peroxiredoxin
PTEN phosphatase and tensin homolog
PTP protein tyrosine phosphatase
R-5-P ribose-5-phosphate
RAS rat sarcoma
ROS reactive oxygen species
RPMI Roswell Park Memorial Institute 1640 medium
Ru-5-P ribulose-5-phosphate
SCF stem cell factor
SOD superoxide dismutase
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SSP serine synthesis pathway
TIGAR TP53-induced glycolysis and apoptosis regulator
TPI triose phosphate isomerase
Trx thioredoxin
TrxR thioredoxin reductase
VAS-2870 3-Benzyl-7-(2-benzoxazolyl) thio-1,2,3-triazolo (4,5-
d) pyrimidine
X-5-P xyulose-5-phosphate

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