Medicine (St Vincent's) - Theses
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ItemDetermining how SRSF2 mutation leads to MDS and CMML and its genetic cooperativities in vivo and in vitro( 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.