Author: Lara Escane
Escane, Lara, 2021 Investigating energy metabolism pathways for therapeutic development and prognostication in chronic lymphocytic leukaemia, Flinders University, College of Medicine and Public Health
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Chronic lymphocytic leukaemia (CLL) is the most common leukaemia of the western world and remains incurable. Uncontrolled proliferation of mature B-lymphocytes results in a compromised immune system and patients often succumb to infection. Despite there being multiple molecular prognostic indicators, one or a combination of these is unable to accurately predict the disease course which varies greatly. Lipoprotein lipase (LPL) expression can be used to predict disease course and implicates the ability of CLL B-lymphocytes to metabolise lipids in disease progression. Additionally, CLL B-lymphocytes have higher intracellular reactive oxygen species (ROS) levels due to highly active mitochondria. We hypothesised that ROS and lipid metabolism are fundamental pathways of CLL pathophysiology and abnormalities in these pathways result in intrinsic resistance of CLL B-lymphocytes to therapy.
In order to test our hypothesis, several objectives were formed.
Firstly, the antioxidant capacity of CLL and healthy B-lymphocytes was assessed with respect to intracellular ROS and cell death to determine if CLL B-lymphocytes from patients with bulky lymphadenopathy (BLA) were better able to handle ROS, preventing ROS-mediated cell death. Individuals with BLA exhibited faster disease progression compared to those without. BLA CLL B-lymphocytes had lower basal intracellular ROS levels, however there was no difference in ROS levels in response to cross-linked RTX therapy. Rotenone caused cell death in CLL B-lymphocytes but not healthy controls implicating an intolerance to the blocking of oxidative phosphorylation and/or increasing intracellular ROS beyond the CLL cell’s capacity to detoxify them.
Secondly, the promoter of the tumour suppressor and antioxidant superoxide dismutase 2 (SOD2) was sequenced to determine if the presence of mutations in transcription factor binding sites that promote SOD2 expression or the introduction of inhibitory transcription factor binding sites are responsible for low SOD2 expression in a pilot cohort of individuals with CLL. The SOD2 promoter was found to be identical to that of the healthy controls. The SOD2 promoter was also assessed for possible suppression by cytosine methylation, however no consistent methylation was detected.
Thirdly, and lastly, lipid metabolic pathways in healthy and CLL B-lymphocytes were assessed in hopes of identifying novel therapeutic targets. CD36/fatty acid translocase was identified as being absent from CLL B-lymphocytes which was unexpected due to the fatty acid-metabolising nature of CLL. RNA expression of fatty acid import genes was assessed, and the tumour suppressor CAV2 and fatty acid transport protein FATP6 were expressed at lower and higher levels in CLL cells compared to healthy controls, respectively. Our fatty acid import inhibition data showed that pinocytosis is the primary mechanism of fatty acid uptake in the CLL B-lymphocyte cell line HG-3. This work was expanded by measuring the fatty acid uptake rate in CLL B-lymphocytes based on their circulatory stage with respect to surface expression of the CXCR4 homing molecule. CLL B-lymphocytes that had recently exited the secondary lymphoid organs had a higher fatty acid uptake rate compared to those homing back to these tissues. Our data suggests that lipids are scavenged in the periphery for storage and use in the proliferative compartment where perfusion is low and hence there is limited nutrient availability.
Our work identified the importance of oxidative phosphorylation and intracellular ROS handling in the survival of CLL B-lymphocytes, particularly those from individuals with BLA which may have a higher antioxidant capacity.
Keywords: chronic lymphocytic leukaemia, reactive oxygen species, fatty acid metabolism, metabolism, methylation, antioxidant, superoxide dismutase 2
Subject: Medical Science thesis
Thesis type: Doctor of Philosophy
Completed: 2021
School: College of Medicine and Public Health
Supervisor: Associate Professor Bryone Kuss