AbstractTo investigate scaffold attachment factor B1 and 2 (SAFB1/2) function in acute lymphoblastic leukaemia (ALL), a transcriptomic differential array screen was interrogated and SAFB1 mRNA expression was found to be significantly lower in T cell ALL (T-ALL) cells compared to normal bone marrow (NBM) cells. Furthermore, the SAFB1/SAFB2 ratio was reduced significantly in both B cell precursor (BCP-) and T-ALL cells. I also found that SAFB2 protein expression was significantly elevated in both ALL subtypes and SAFB1 overexpression induced apoptosis within 48 hours in ALL cells. In contrast, apoptosis was not induced in NBM cells and fibroblasts following SAFB1 overexpression. These data suggest decreased SAFB1 protein expression may promote oncogenesis and overexpressing SAFB1 may form the basis for gene therapy strategies.
The stress response (as characterised by the induction of heat shock factor 1 (HSF1), heat shock proteins (HSPs) and formation of nuclear stress bodies (nSBs) and action of anti-cancer HSP90 inhibitors (celastrol and 17-DMAG) has been characterised in ALL cells. Experiments to characterise the ALL stress response found that HSF1 was constitutively overexpressed and HSF1 nuclear border expression was present in primary BCP-ALL and T-ALL cells following heat shock and 17-DMAG treatment compared with NBM. SAFB1/2 co-localises with HSF1 following a HS in HeLa cells and primary fibroblasts. However, neither SAFB1 nor SAFB2 was found to co-localise with HSF1 following a HS in ALL cells. These findings suggest the stress response differs in ALL cells and may render them more permissive to treatment with HSP90 inhibitors.
RGG/RG and SUMOylation mutants of SAFB1/2 were made to investigate the importance of post translational modifications (PTMs) in regulating SAFB1/2 interactions. Experiments revealed that SAFB1 SUMOylation mutants had a significantly increased interaction with the known oncogene, serine/arginine-rich splicing factor 1 (SRSF1), following heat shock. Results also showed that interactions between SAFB2 delta-RGG/RG methylation mutants and SRSF1 was reduced compared with wild-type SAFB2 under basal and heat shock conditions. Proteomic analyses of SAFB1/2 interactions were undertaken using Tandem Mass Tag Mass Spectrometry (TMT-MS) and bioinformatic analyses suggested they regulate RNA processing, splicing, transcription and translation. Interestingly SAFB2 bound more proteins than SAFB1 in T-ALL cells but bound significantly fewer proteins in HeLa cell. Together, these data highlight the pivotal role of SAFB1/2 play in regulating critical cellular processes and altered indicate SAFB1/2 expression ratios could be important in promoting tumorigenicity.
|Date of Award||24 Mar 2020|
|Supervisor||James B Uney (Supervisor) & Allison Blair (Supervisor)|