AbstractAcute myeloid leukaemia (AML) is a genetically heterogeneous disease characterised by the expansion of immature blast cells in the bone marrow and it primarily affects individuals aged 65 and over. Through next generation sequencing studies, the genetic landscape of AML has become increasingly better characterised leading to the identification of recurrent gene mutations. Cohesin complex genes were discovered to be mutated in 13% of AML patients and STAG2 was the most commonly mutated subunit (6%), however it is also the most understudied. The Cohesin complex is essential for sister chromatid cohesion as well as organizing the 3D genome, controlling gene expression and DNA repair. The consequences of a STAG2 mutation on Cohesin function is not well understood and in particular, the effects on DNA damage repair remain to be fully elucidated. In addition, it is likely that the epigenome is affected by Cohesin’s involvement in chromatin compaction and transcriptional regulation, however, this has not previously been explored in the literature and how a STAG2 mutation would impact the epigenome is unknown. Therefore, the purpose of this study was to determine how a STAG2 mutation affects DNA repair, gene expression and DNA methylation.
A STAG2 knockout (ΔSTAG2) isogenic cell line model was generated by my colleague Dr James Smith in the OCI-AML3 parental line and it was used throughout this project. To explore the impact on DNA repair immunofluorescence (IF) assays were performed staining 53BP1 and RAD51 in the isogenic cells post-irradiation. This data confirmed that double stranded break (DSB) repair is impaired in ΔSTAG2 cells and excitingly, this conferred PARP inhibitor sensitivity. To further investigate the potential for therapeutic targeting of ΔSTAG2 the isogenic cells were screened against Selleckchem’s DNA Damaging Compounds Library identifying 32 compounds to which mutant cells were significantly more sensitive. Importantly, these results highlight the opportunity for targeted therapy in AML patients with a STAG2 mutation.
Expanding on these findings DNA fibre assays were performed which unearthed an essential role for STAG2 at the replication fork and revealed that ΔSTAG2 cells have an intra-S phase checkpoint defect. Therefore, this could be the reason why ΔSTAG2 cells were significantly more sensitive than WT to inhibitors of DNA synthesis and topoisomerase in the aforementioned drug screen. The transcriptomic consequences of STAG2 depletion were investigated by RNA
sequencing revealing a modest impact on gene expression with 1,899 significantly differentially expressed genes (DEGs) in mutant cells relative to WT. Enrichment analysis confirmed these DEGs were enriched in DNA repair, DNA replication and cell cycle pathways further validating the data presented earlier in this thesis. Whole genome bisulfite sequencing of the isogenic cells uncovered altered global DNA methylation caused by chronic STAG2 loss. Approximately 20% of differential methylation of ΔSTAG2 cells relative to WT occurred at CpG loci of which 88% were hypermethylated, synonymous with repressed gene expression. Thus, this is in concordance with RNA sequencing data which showed most DEGs were downregulated.
In conclusion, this project has demonstrated that in an AML model a STAG2 mutation causes DNA repair and cell cycle checkpoint defects conferring PARP inhibitor sensitivity. Significantly, this highlights the potential of using a STAG2 mutation as a biomarker for response to Olaparib and Talazoparib in AML. Additionally, NGS studies revealed modest transcriptomic changes accompanied by altered global DNA methylation in cells depleted of STAG2. Excitingly, enrichment analysis of DEGs and DMRs revealed many novel and some unexpected affected pathways that could be leukaemogenic and therefore offer avenues for future investigation.
|Date of Award||Jul 2021|
|Sponsors||Leukaemia & Lymphoma NI|
|Supervisor||Ken Mills (Supervisor), Christopher Scott (Supervisor) & Adone Tielenius Kruythoff-Mohd Sarip (Supervisor)|
- Acute myeloid leukaemia (AML)
- DNA damage repair
- PARP inhibition
- cell cycle checkpoints