Characterisation of the BCAT1 CXXC motif in Acute Myeloid Leukaemia

Hillier, James (2020) Characterisation of the BCAT1 CXXC motif in Acute Myeloid Leukaemia. PhD thesis, University of Worcester.

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Abstract

Acute Myeloid Leukaemia (AML) is the most common acute white blood cell
cancer in adults, with a U.K. incidence of 3,100 new cases/year approximately 30% of
all leukaemia. Survival remains below 15% for patients over the age of 60 diagnosed
with AML, who respond poorly to standard non-specific chemotherapy treatment,
therefore there is a need for new targeted therapies .
Recently, expression of cytosolic isoform of Branched Chain Aminotransferase
(BCAT1) has been correlated with shorter overall survival in AML. Current research has
focussed on the BCAT1 metabolites α-ketoglutarate (α-KG) , glutamate and leucine,
however BCAT1 contains a redox active CXXC motif, a feature of antioxidant enzymes
such as Thioredoxin, suggesting BCAT1 may have a novel antioxidant role.
Overproduction of Reactive Oxygen Species (ROS) in AML is extremely well
characterised and contributes to the pathogenesis of the disease. Targeting leukaemia
cells with pro-oxidants to overwhelm the antioxidant capacity of the cell is recognised
as an attractive strategy, particularly in respect to leukemic stem cells which are a major
cause of therapy resistance and relapse. Therefore, in this study, the potential
antioxidant effect of the BCAT1 CXXC motif was investigated with respect to well
documented redox mediated processes dysregulated in AML. These redox mediated
processes included cellular proliferation, differentiation and apoptosis. To evaluate this
a myeloid leukaemia cell line, U937 (ATCC CRL 1293.2) was engineered to stably
overexpress wild-type BCAT1 protein and CXXC motif mutant BCAT1.
The key findings of the study showed that the BCAT1 CXXC motif provides a
protective effect against ROS mediated apoptosis induced by serum starvation in U937
cells. Moreover the CXXC motif reduced intracellular ROS and CD36+
cells, a marker of
macrophage differentiation following addition of Phorbol 12-myristate 13-acetate
(PMA) which has been demonstrated to induce differentiation of U937 cells using H202,
a biologically relevant ROS species, as a secondary messenger. Finally, the feasibility of
targeting BCAT1 with standard chemotherapy, metabolic inhibitors and pro-oxidants
therapy has been explored with a view to developing a treatment strategy for patients
with BCAT1high and BCAT1low levels of expression. For the first time this study
demonstrated that the BCAT1 CXXC motif reduces intracellular ROS and provides
protection against apoptosis in response to treatment with pro-oxidant Rotenone whilst
BCAT1 increases sensitivity to standard chemotherapeutic Cytarabine (Ara-C).
Overall, the findings in this study indicate that the BCAT1 CXXC motif acts as a novel
antioxidant in AML impacting redox mediated cellular differentiation and apoptosis
through ROS modulation. Similarly, the BCAT1 and the antioxidant capacity of the CXXC
motif can protect against pro-oxidant therapy, an important finding that could inform
treatment strategies in future.

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