Medicine (St Vincent's) - Theses

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    The roles of retinoic acid receptors and target gene HOXA1 in regulating haematopoiesis and myelodysplastic syndromes
    Grace, Clea Simes ( 2022)
    Haematopoietic stem cells (HSCs) are multipotent cells responsible for the maintenance of the haematopoietic system throughout life. Dysregulation of the balance between HSC self-renewal, death and differentiation can have serious consequences such as myelodysplastic syndromes (MDS) or leukaemia. All-trans retinoic acid (ATRA), the biologically active metabolite of retinoic acid (RA) has pleiotropic effects on haematopoietic cells, maintaining haematopoiesis via complex intrinsic and extrinsic mechanisms involving retinoic acid receptor-alpha (RARalpha) and RARgamma signalling. Conditional murine Rar knockout models were utilised to determine the intrinsic and extrinsic impacts of Rara and/or Rarg deletion on HSCs and their progeny in vivo via global, postnatal deletion, haematopoietic cell-specific deletion (whole bone marrow transplants (WBMTs)) and HSC and progenitor-specific deletion (Lineage-c-Kit+Sca-1+ (LKS+) WBMTs). These data revealed novel roles of Rara in haematopoiesis, with postnatal deletion resulting in intrinsically-mediated myeloid-biased haematopoiesis, reduced numbers of CD8+ T cells and altered HSC reconstitution capacity, in addition to extrinsically-mediated aberrant BM erythropoiesis and B lymphopoiesis. Loss of either Rara and/or Rarg resulted in myeloid-biased haematopoiesis and concurrent deletion (expression of Rarb only) enhanced HSC exhaustion. Contrary to germline Rarg-/- studies, postnatal Rarg deletion had a limited impact on haematopoiesis. The observed phenotypical differences between germline and global, postnatal Rar deletions may be due to a combination of developmental and microenvironmental interactions, interactions between Rara and Rarg and their differential target genes and compensatory effects. Whilst the relative contribution of RA signalling to its haematopoietic regulation is unknown, homeoboxa1 (HOXA1) is an RARgamma target gene and its full-length isoform (HOXA1-FL) is frequently upregulated in human MDS. A series of studies utilising conditional murine haematopoietic Hoxa1 knockin mice revealed that whilst recipient mice developed MDS during serial transplantation, consistent with previous studies, the levels of Hoxa1-FL overexpression were insufficient to result in progression to secondary acute myeloid leukaemia (sAML). Additionally, Hoxa1 overexpression resulted in profound, persistent and serially transplantable macrothrombocytopenia despite a depleted yet highly megakaryocyte-primed HSC and progenitor compartment. The macrothrombocytopenia resulted from ineffective thrombopoiesis by dysfunctional, low ploidy megakaryocytes, potentially due to inflammation-mediated lineage skewing and/or late-stage developmental defect(s). Dysregulated BM erythropoiesis and granulopoiesis were also observed. These phenotypes were consistently evident early, persisted in non-transplanted pre-malignant mice and became more profound during serial transplantation and after MDS progression. Phenotypic severity also depended on Hoxa1 gene-dosage and isoform expression. Furthermore, the megakaryocyte-priming correlated to increased DNA damage and/or deficient DNA damage responses (DDR), increased expression of inflammatory markers and altered programmed cell death, suggesting that an altered inflammatory microenvironment and associated haematopoietic lineage skewing may have contributed to the Hoxa1-FL-mediated haematopoietic defects, although this remains under investigation. Whilst the precise mechanisms underlying these phenotypes therefore remain to elucidated, it is evident that Rara, Rarg and Hoxa1-FL have pleiotropic impacts on haematopoiesis that are both cell-type and maturation-stage dependent. Elucidation of these effects in murine models are critical steps in the development of targeted therapies for MDS patients with increased Hoxa1-FL expression and for other haematopoietic malignancies to which dysregulated Hoxa1 and/or RA signalling may contribute.
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    Determining how SRSF2 mutation leads to MDS and CMML and its genetic cooperativities in vivo and in vitro
    Xu, Jane Jialu ( 2020)
    Myelodysplastic syndromes (MDS) are a group of heterogeneous clonal stem cell malignancies characterised by multilineage cytopenia and dysplasia. MDS mostly occurs in aged populations where there are limited therapeutic options. Compared to MDS, chronic myelomonocytic leukaemia (CMML) presents with a monocytosis feature and has a poor survival of 16 months for high-risk patients. In the past decade, several sequencing studies have defined the complex molecular landscapes of MDS and CMML. SRSF2, a component of RNA splicing machinery, is one of the most frequent mutations in MDS and CMML. To understand the consequences and effects of SRSF2P95H mutation on normal haematopoiesis, several groups, including our lab, have generated in vivo mouse models using various gene targeting strategies and Cre recombinases. These models demonstrated some effects of SRSF2P95H mutation on haematopoiesis, and described certain mis-splicing changes in the Srsf2P95H/+ cells. However, the mechanism and role of SRSF2P95H mutation in promoting and initiating MDS/CMML are still poorly defined. My thesis aimed to address key knowledge gaps by examining the cell of origin, transcriptomic/splicing changes, synthetic lethal genetic interactions, and co-operative interactions of SRSF2P95H/+ mutation in the initiation of MDS/CMML. In the first part of my thesis, I assessed the cell of origin in SRSF2P95H MDS by characterizing a conditional knock-in Srsf2P95H/+ mouse model, using LysM-Cre. After activating Srsf2P95H/+ mutation in myeloid progenitors, I observed no development of MDS even after prolonged ageing (up to 52 weeks) and only mild changes in haematopoiesis. Compared to the stem cell activation model (hScl-CreERT2) that we reported, the results of LysM-Cre demonstrated that a myeloid progenitor is not the cell of origin in SRSF2mut MDS. In the second part of my thesis, I analyzed the transcriptomic and splicing changes of Srsf2P95H/+ cells, using both purified stem and progenitor cell populations as well as Hoxb8 immortalized cell lines. The transcriptome analysis revealed up- and down-regulation of lineage associated genes and up-regulation of MDS associated pathways and the p38 MAPK kinase pathway. The splicing analysis demonstrated skipped exons as the most frequent alternative splicing event. In terms of specific mis-splicing targets, I examined exon inclusion in several reported transcripts and compared the most frequently mis-spliced genes across 12 human SRSF2mut and murine Srsf2mut datasets. Through this analysis, I found that mRNA processing and DNA repair represent the top mis-spliced pathways in Srsf2P95H/+ cells. I also present a pilot study of single cell RNA sequencing of Srsf2P95H/+ stem and primitive progenitor populations, which unveiled a myeloid-biased signature and enhanced myeloid differentiation of the Srsf2P95H/+ stem cells. In the third part of my thesis, I explored the synthetic lethality of Srsf2P95H/+ cells with a pooled CRISPR knock-out screen. I discovered that loss of DNA repair or cell cycle pathways was synthetic lethal with Srsf2P95H/+ mutation. Consistent with this genetic lethality, I demonstrated that Palbociclib, a CDK6 inhibitor, could preferentially target the Srsf2P95H/+ cells. This finding opens up new therapeutic windows beyond known spliceosome inhibitors for SRSF2mut MDS. In the fourth and last part of my thesis, I generated and characterized two multi-genic mutation models: Srsf2P95H/+ Tet2-/- and Srsf2P95H/+ Cbl-/-. In the Srsf2P95H/+ Tet2-/- model, I observed profound myeloid bias, B lymphoid suppression and increased ST-HSC percentages in the stem cell compartment after 52 weeks of activating/deleting mutations in the haematopoietic stem cells. Within the Srsf2P95H/+ Tet2-/- cohort, I also observed development of CMML in both native haematopoiesis and transplantation settings. So far, this is the first model to demonstrate synergistic interactions between Srsf2P95H/+ and Tet2-/- mutation, as well as initiation of CMML in vivo. For the Srsf2P95H/+ Cbl-/- model, I characterized a small cohort of mice due to prolonged breeding difficulties. Nevertheless, I discovered increased myeloid proliferation in the double Srsf2P95H/+ Cbl-/- and Srsf2P95H/+ Cbl+/- mutants. Collectively, the work included in this thesis creates an original contribution to understanding the role of SRSF2P95H mutation in MDS, its synthetic lethal genetic interactions, potential therapeutic targeting of SRSF2P95H cells, and how it co-operates with other recurrent mutations in initiation of CMML.