Targeting reactive oxygen species for the treatment of acute myeloid leukaemia

Germon, Zacary P.

Title: Targeting reactive oxygen species for the treatment of acute myeloid leukaemia
Creator: Germon, Zacary P.
Relation: University of Newcastle Research Higher Degree Thesis
Resource Type: thesis
Date: 2022
Description: Research Doctorate - Doctor of Philosophy (PhD)
Description: Historically, reactive oxygen species (ROS) were considered harmful biproducts of cellular metabolism. Now, it is well established that ROS are functionally important molecules that play a diverse range of roles in a variety of physiological and pathological contexts, where they are tightly regulated to maintain what is referred to as redox homeostasis. ROS are crucial regulators of cellular signalling via their ability to reversibly (and irreversibly) oxidise cysteine residues, influencing intra and inter-cellular protein interactions, and therefore, function. Many diseases, including cancer have now been confirmed as being associated with increased production of ROS or a disruption to redox homeostasis. Acute myeloid leukemia (AML) is no exception, where increased ROS is now considered a hallmark of the disease. AML is the most common aggressive leukaemia, with a 5-year survival rate for adults of 30% and 67% for children. Annually in Australia, 950 patients are diagnosed with AML, with 800 patients unfortunately succumbing to their disease. Activating mutations to the receptor tyrosine kinase ‘FLT3’ are the most recurrent mutations, seen in 20-30% of patients. Notably, the most common form of mutation to FLT3 (Internal tandem duplication or ITD) are associated with increased ROS production, a link that is of significant clinical interest given that ROS have been shown to promote genomic instability through the oxidation of DNA bases, driving clonal evolution, treatment resistance and poor outcomes. These are all major hurdles in the pursuit of improved outcomes for AML patients, and whilst the development of FLT3 inhibitors have led to a clinical benefit in FLT3 mutant AML patients, relapse rates and long-term survival remains poor, highlighting the need for improved treatment strategies. Hence, the overarching aim of this thesis is to determine the therapeutic potential of targeting ROS for the improved treatment of AML. In the first data chapter of this thesis (Chapter 4), we utilise novel mass spectrometry-based proteomics incorporating global assessment of redox driven oxidative posttranslational modifications, termed ‘oxPTMs’, in tandem with phosphoproteomic profiling of AML primary patient biopsy samples (taken from the bone marrow at diagnosis). This formed the first proteome wide assessment of redox signalling in AML and provides site-specific characterisation of the functional impact of ROS, with our data showing significant increases in oxPTMs in ITD mutant samples when compared to FLT3-wild-type samples. Our data also reveals the ROS producing NADPH oxidase 2 (NOX2) and all associated subunits to be present across all samples, revealing a relevant therapeutic target. Importantly, targeting NOX2 (inhibitors/siRNA) was synergistically cytotoxic when combined with FLT3-inhibitors. Inhibition of NOX2 in patient derived AML xenograft models decreased leukaemia markers in the blood (huCD45+) and significantly increased survival as a monotherapy in the FLT3-ITD+ cohort. NOX2-inhibitors combined with FLT3-inhibitors in a FLT3-ITD xenograft model significantly increased overall survival compared to monotherapeutic strategies. Mechanistic studies using redox proteomics on AML cells harvested from the bone-marrow of xenograft models revealed NOX2 inhibition decreased total NOX2 expression, leading to decreased oxidation of FLT3-C828, inhibiting downstream signalling. Subsequent analysis of signalling pathways downstream of FLT3 revealed decreased activity of the key growth and survival factors STAT5 and ERK, known to contribute to leukaemogenesis. Following on from these findings we reveal a link between oxidative stress and epigenetic reprogramming, via reversible oxPTMs identified in the DNA binding domain of the methyltransferase enzyme DNMT1 (C686) (Chapter 6). We show that oxidation of DNMT1 reduces its ability to methylate DNA, and, that pharmacological inhibition of NOX2 (in patient derived xenograft models of FLT3-ITD AML) reduced DNMT1 oxidation at C686. Importantly, targeting NOX2-modulated global patterns of DNA methylation, including CpG islands of protein kinase B (AKT) oncogene and critical regulators of the PI3K/AKT pathway. We then demonstrate a synergistic combination between NOX2 inhibitors and the hypomethylating agent 5-Azacytadine in human AML cells, a clinically relevant combination that warrants further investigation. Overall, this body of work provides extensive evidence for the cooperation of redox signalling with oncogenic kinases and epigenetic regulatory machinery, highlighting the potential of targeting ROS production for the treatment of AML.
Subject: leukaemia; cell signalling; proteomics; targeted therapies; reactive oxygen species
Identifier: http://hdl.handle.net/1959.13/1513821
Identifier: uon:56772
Language: eng

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