Investigating the role of CEBPA & MYBL2 in haematological disorders

Myelodysplastic syndromes (MDS) are a diverse group of clonal haematological disorders that affect the proliferation and differentiation of haematopoietic stem cells, in which the bone marrow fails to produce mature blood cells. There is no cure for MDS; more than half of patients with MDS do not respond to current treatments, and 40% of MDS cases progress to acute myeloid leukaemia (AML). Consequently, there is an urgent clinical need to understand the mechanisms underlying these blood disorders to develop effective diagnostic and therapeutic strategies. Through this thesis, we studied the contribution of two transcription factors, C/EBPα and MYBL2, to the MDS disease phenotype. The CEBPA gene encodes the C/EBPα protein, a transcription factor required for haematopoietic stem cell self-renewal and granulocytic commitment. 8% of patients with MDS have mutations in CEBPA at the time of initial diagnosis, and 12% progress from MDS to AML. The main goal of this study is to understand the contribution of the CEBPA mutation which disrupts the C-terminal domain to the dysplastic phenotype observed in MDS. To accomplish this, we developed a novel in vitro model system that combines human induced pluripotent stem cells (hiPSCs) with CRISPR-Cas9 technology to produce a heterozygous mutation in CEBPA, which results in the disruption of the DNA binding domain. Our data show that the hiPSC-CEBPA$^{+/mut}$ could differentiate into hematopoietic stem/progenitor cells (HSPCs) (CD34$^+$/CD43$^+$, CD34$^+$/CD45$^+$). HSPCs generated from hiPSC-CEBPA$^{+/mut}$ clones differentiated in methylcellulose semi-solid media, albeit at a reduced capacity compared to isogenic control iPSCs. Moreover, consistent with previous studies showing the importance of CEBPA in regulating myeloid differentiation, HSPCs derived from hiPSC-CEBPA$^{+/mut}$ showed an inability to form granulocytic colony-forming units (CFU-G); they displayed altered expression of myeloid differentiation-required genes, such as PU.1, GATA2, and RUNX1, and exhibited a high percentage of aberrant myeloid cells indicated by the presence of pseudo Pelger-Huët anomaly. HSPCs derived from hiPSC- CEBPA$^{+/mut}$ promoted aberrant erythroid differentiation, as evidenced by the elevated expression of EPO-R and TFRC and the presence of aberrant morphology, such as multi-nucleated erythroblasts. The other transcription factor studied in this thesis is MYBL2, a gene located in the long arm of chromosome 20, commonly deleted in 5-10% of MDS and AML patients (del20q). MYBL2 is essential for sustaining the balance between self-renewal and the differentiation of haemopoietic stem cells (HSCs). In MDS, MYBL2 has been found to play a tumour suppressor role. Studies have shown that half of patients with MDS have low levels of MYBL2, regardless of del20q cytogenetic abnormalities, indicating that dysregulation of MYBL2 could significantly affect MDS progression. Attempts to generate hiPSCs with low levels of MYBL2 using CRISPR-Cas9 technology failed; thus, we generated three different human leukaemia cell lines to downregulate MYBL2 by lentiviral transduction with doxycycline-inducible MYBL2shRNA. After generating and validating these MYBL2 and isogenic control cell lines, we studied their erythroid lineage differentiation capacity after cytarabine (Ara-C) treatment. The results showed that reducing MYBL2 in leukaemia cell lines did not affect cell numbers or differentiation when treated with 1 µM Ara-C to drive erythroid differentiation. Our work using different experimental systems highlights the advantages of hiPSCs in studying the role of these transcription factors to elucidate the molecular mechanisms underlying the initiation and progression of haematological disorders

Crosstalk between AML and stromal cells triggers acetate secretion through the metabolic rewiring of stromal cells

Acute myeloid leukaemia (AML) cells interact and modulate components of their surrounding microenvironment into their own benefit. Stromal cells have been shown to support AML survival and progression through various mechanisms. Nonetheless, whether AML cells could establish beneficial metabolic interactions with stromal cells is underexplored. By using a combination of human AML cell lines and AML patient samples together with mouse stromal cells and a MLL-AF9 mouse model, here we identify a novel metabolic crosstalk between AML and stromal cells where AML cells prompt stromal cells to secrete acetate for their own consumption to feed the tricarboxylic acid cycle (TCA) and lipid biosynthesis. By performing transcriptome analysis and tracer-based metabolic NMR analysis, we observe that stromal cells present a higher rate of glycolysis when co-cultured with AML cells. We also find that acetate in stromal cells is derived from pyruvate via chemical conversion under the influence of reactive oxygen species (ROS) following ROS transfer from AML to stromal cells via gap junctions. Overall, we present a unique metabolic communication between AML and stromal cells and propose two different molecular targets, ACSS2 and gap junctions, that could potentially be exploited for adjuvant therapy

Crosstalk between AML and stromal cells triggers acetate secretion through the metabolic rewiring of stromal cells

Acute myeloid leukaemia (AML) cells interact and modulate components of their surrounding microenvironment into their own benefit. Stromal cells have been shown to support AML survival and progression through various mechanisms. Nonetheless, whether AML cells could establish beneficial metabolic interactions with stromal cells is underexplored. By using a combination of human AML cell lines and AML patient samples together with mouse stromal cells and a MLL-AF9 mouse model, here we identify a novel metabolic crosstalk between AML and stromal cells where AML cells prompt stromal cells to secrete acetate for their own consumption to feed the tricarboxylic acid cycle (TCA) and lipid biosynthesis. By performing transcriptome analysis and tracer-based metabolic NMR analysis, we observe that stromal cells present a higher rate of glycolysis when co-cultured with AML cells. We also find that acetate in stromal cells is derived from pyruvate via chemical conversion under the influence of reactive oxygen species (ROS) following ROS transfer from AML to stromal cells via gap junctions. Overall, we present a unique metabolic communication between AML and stromal cells and propose two different molecular targets, ACSS2 and gap junctions, that could potentially be exploited for adjuvant therapy