Characterisation and Preferential Targeting of Leukaemic Stem Cells in Acute Myeloid Leukaemia

Abstract

Failure to completely eradicate leukaemic stem cells (LSCs) in acute myeloid leukaemia (AML) is believed to contribute to the high incidence of relapsed and refractory disease. However, therapeutic targeting of LSCs remains a major challenge due to their phenotypic and functional heterogeneity, and their reliance on the bone marrow microenvironment. Despite these caveats, the CD34+CD38dim sub-population within the KG1a AML cell line remains a commonly used model to study ‘LSC-like’ cells in vitro. This thesis describes the detailed characterisation of a sub-population within the CD34+CD38dim ‘LSC-like’ compartment of KG1a cells with reduced expression of C-type lectin-like molecule 1 (CLL-1). Using cell sorting-based approaches, CD34+CD38dimCLL-1 dim cells were shown to display functional characteristics typically associated with LSCs, including relative quiescence, low intracellular reactive oxygen species (ROS) levels, enhanced clonogenicity and an increased capacity for self-renewal. Importantly, this CD34+CD38dimCLL-1 dim population was also identified in primary AML samples and was shown to exhibit similar functional characteristics. Intriguingly, bi-directional plasticity in both CD38 and CLL-1 antigen expression was observed in purified KG1a cells, whereby the CD34+CD38dimCLL-1 dim ‘LSC-like’ cells possessed the ability to become CD34+CD38brightCLL-1 bright, and CD34+CD38brightCLL-1 bright ‘AML blast-like’ cells possessed the ability to become CD34+CD38dimCLL-1 dim. This challenged the conventional clonal hierarchical model of LSCs, but it remains to be determined whether primary AML cells can also demonstrate this plasticity. Assessment of the relative potency of the AML chemotherapy drug cytarabine (AraC) in the putative CD34+CD38dimCLL-1 dim ‘LSC-like’ cells versus the CD34+CD38brightCLL-1 bright ‘AML blast like’ cells showed that primary CD34+CD38dimCLL-1 dim cells were relatively resistant (a phenomenon typically associated with LSCs). In contrast, the CD34+CD38dimCLL-1 dim ‘LSC-like’ cells appeared to show increased sensitivity in the KG1a cell line. However, it remains unclear whether this apparent dichotomy represents a fundamental difference between KG1a cells and primary AML cells or whether the phenotypic plasticity observed in KG1a cells is promoted by exposure to AraC. Either way, these results highlight the complex, heterogeneous nature of AML. Given the acknowledged importance of the bone marrow microenvironment in the maintenance of LSCs, the effect of hypoxia on the phenotype and function of KG1a cells was explored. Hypoxia enhanced LSC characteristics; a phenomenon caused, at least in part, by hypoxia-mediated changes in gene transcription. Gene expression analysis showed that the KG1a CD34+CD38dimCLL-1 dim ‘LSC-like’ and CD34+CD38brightCLL-1 bright ‘AML blast-like’ cells were transcriptionally distinct under both normoxic and hypoxic conditions and identified NFκB targeting through proteasome inhibition as a potential mechanism to therapeutically deplete the ‘LSC like’ cells.

Date of Award	Jan 2024
Original language	English
Awarding Institution	University of Brighton
Supervisor	Chris Pepper (Supervisor) & Andrea Pepper (Supervisor)