Establishing models and investigating age-relevant disease biology and biomarkers in MLL rearranged acute myeloid leukaemia

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Saudi Digital Library
Acute myeloid leukaemia (AML) is a clinically, biologically, and genetically heterogeneous disease. AML frequently appears in adults; however, it makes up 20% of paediatric leukaemia with distinct disease identity. Currently, treatment for paediatric AML has been extrapolated from adult AML treatment, which suggests that difference in disease biology between children and adults is absent. In fact, more than 40% of children treated with standard chemotherapy experience a high relapse rate, and Mixed Lineage Leukaemia (MLL) rearrangements are most commonly seen in relapse paediatric patients. Thus, there is an urgent need to understand the biology of paediatric AML which might enable age specific treatment approaches. Previously, our lab has demonstrated that the CD180 gene is positively enriched in the molecular profile of paediatric compared to old AML. Another study reported that CD180 cell surface expression increased to 60-fold in AML leukaemia stem cells (LSC) compared to normal haematopoietic stem cells (HSCs). CD180 is known as a pharmacodynamic biomarker for Bromodomain and Extra-Terminal motif (BET) inhibition. Thus, we hypothesised that CD180 could identify paediatric AML (PAML) entity or provide more understanding of the genes (biomarker) and pathways implicated in PAML. We first aimed to demonstrate the CD180 expression pattern and effect of BET inhibitor (BETi) in AML. We sought to investigate CD180 mRNA expression levels and CD180 cell surface expression in paediatric and adult AML cell lines. Interestingly, CD180 mRNA expression was high in all MLLr cell lines regardless of age. CD180 cell surface expression was higher in the majority of PAML samples compared to adult AML primary samples. We found that the bone marrow microenvironment (BMM) induced CD180 overexpression in MLLr cell lines. Downregulation of CD180 associated with increased apoptosis was achieved when MLLr cells were treated with the BETi; AZD5153 in co-culture (CC). The cell viability was reduced significantly when the cells were treated with AZD5153 in CC compared to liquid culture (LC). We have also shown that Cytarabine (Ara-C) surviving cells overexpress CD180 in all AML cell lines regardless of cytogenetic abnormalities. We used BETi to reduce the viability of AML cells that express high levels of CD180. Thus, CD180 expression is upregulated on AML cells upon different stimuli. BMM upregulates CD180 expression exclusively in MLLr cell lines, but chemotherapy causes CD180 overexpression regardless of cytogenetic alterations. Next, we want to investigate the transcriptional profile of CD180hi cells in MLLr cell lines (ThP-1 and Molm-13) and its biological significance in MLLr AML. We found that the top ten differentially expressed genes (DEGs) were associated with metabolism, migration, cell cycle, and cell to cell adhesion. Interestingly, gene set enrichment analysis (GSEA) showed that CD180hi cells were significantly enriched with pathways associated with BMM; such as metastasis, cell migration, and metabolisms; such as nucleotide synthesis, fatty acid and carbohydrate metabolism, and poor survival. Connectivity map (CMap) analysis was used to find a potential therapeutic tool to target CD180hi population, whereby, 30 drug candidates were identified whose effect may reverse the CD180hi signatures. One of these drugs was BETi, which we used to downregulate CD180 expression. Within this thesis, we have linked CD180 expression with MLLr AML, and We want to investigate the CD180 expression pattern and its significance in paediatric MLLr AML. Therefore, we generated a novel haematopoietic inducible MLL-AF9 model in prenatal (E0), postnatal (P0), and adult (> 20 weeks) to recapitulate the disease in infants, neonates, and adults. We successfully obtained different types of leukaemia when MLL-AF9 was induced at P0, and the phenotypes were acute myelomonocytic leukaemia (AML-M4), acute megakaryoblastic leukaemia (AML-M7), and mixed phenotype acute leukaemia (MPAL). However, when MLL-AF9 was induced prenatally or in adult age, most of the mice developed AML exclusively, and some of the mice did not show any signs of illness for 1 year after oncogene induction. MLL-AF9 mRNA expression levels were comparable in haematopoietic stem and progenitor cells (HSPCs) except the granulocyte-monocyte progenitors (GMPs) across all the cohorts. On the other hand, MLL-AF9 expression levels was higher in GMPs compared to HSCs from postnatal model and this was accompanied by GMP expansion in the bone marrow (BM). The AML cells from double transgenic (DTG) mice showed the ability to self-renew in vitro across all the cohort. BM of adult diseased mice cause lethal transplantable AML with short latency, fully penetrant, and extramedullary infiltration in vivo. This model will be utilised further to define the paediatric leukaemia stem cells, to characterise CD180 expression patterns in paediatric leukaemia stem cells (LSCs) compared to adult LSCs, and to test the significance of CD180 as a potential paediatric LSC marker. The work presented in this thesis presents a novel gene signature for CD180hi cells that exhibits LSC characteristics in MLLr-AML, and it can be targeted with BETi and Kinase inhibitors. Finally, MLL-AF9 model can be an experimental tool to investigate age-specific biomarkers; for instance CD180, and targeted treatment strategies for paediatrics and adults.
Establishing models and investigating age-relevant disease biology and biomarkers in MLL rearranged acute myeloid leukaemia