- Title
- Targeting PP2A activation as a novel therapeutic strategy for receptor tyrosine kinase driven leukaemia
- Creator
- Smith, Amanda Melanie
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2014
- Description
- Research Doctorate - Doctorate of Philosophy (PhD)
- Description
- The receptor tyrosine kinases (RTK) c-KIT and FLT3 are overexpressed or mutated in acute myeloid leukaemia (AML) and numerous other cancers. Constitutive activation of these RTKs stimulates downstream kinase signalling cascades including PI3K/Akt, Ras/MAPK, and JAK/STAT, which lead to increased proliferation and leukaemogenesis. Although inhibition of c-KIT with imatinib mesylate (IM) has been successful in treating gastrointestinal stromal tumour (GIST) patients, responses in AML patients have been less favourable, largely due to intrinsically resistant kinase domain mutations. There are currently no clinically available, therapeutic compounds targeting c-KIT or FLT3 in AML. Considering around half of core-binding factor AML express c-KIT mutations and one third of all AML patients express FLT3 mutations, a greater understanding of the signalling pathways regulated by these RTKs is necessary to identify novel targets for improved therapy of these leukaemias. Protein phosphatase 2A (PP2A) is a fundamental cellular signalling molecule that regulates many of the signalling pathways deregulated in leukaemia. PP2A is a heterotrimeric enzyme composed of a catalytic (PP2A-C), scaffolding (PP2A-A) and one of a number of regulatory (PP2A-B) subunits. PP2A is a proposed tumour suppressor, regulated by interaction with regulatory B subunits, or one of a number of endogenous PP2A interacting proteins. The role of functional PP2A inactivation has recently been recognised as a mediator of oncogenic tyrosine kinase driven leukaemia. This thesis is the first to examine the role of PP2A inhibition in c-KIT and FLT3 driven leukaemogenesis. Utilising the FDCP.1 mouse myeloid cell line expressing imatinib-sensitive (V560G) or –resistant (D816V) mutant c-KIT, and the BaF3 murine lymphoblastic cell line expressing constitutively active FLT3-ITD (FLT3 with internal tandem duplication), I show for the first time that constitutive activation of c-KIT or FLT3 induces functional inactivation of PP2A tumour suppressive activity. Importantly, pharmacological reactivation of PP2A by FTY720 or AAL(S) was found to inhibit the growth and survival of cells expressing these mutant RTKs. Furthermore, I have shown that FTY720 mediated activation of PP2A induces apoptosis of AML patient blasts expressing FLT3 mutations, compared to patient cells expressing the WT FLT3 receptor. PP2A inhibition was found to be associated with reduced protein, but not mRNA, expression of PP2A structural and regulatory subunits, and this is functionally important as over-expression of exogenous PP2A-A induced apoptosis in these cells. PP2A inhibition was further associated with the expression of novel variants of the endogenous PP2A inhibitory protein, SET. These novel variants were also identified in AML patient blasts. Further investigations showed that these novel variants alter the subcellular localisation of SET, and its co-localisation with PP2A. Investigations into the mechanism of action of the PP2A activating compound, FTY720, found that FTY720 alters the expression and sub-cellular location of these novel SET proteins, suggesting that they are functionally important in the cellular response to FTY720. Finally, I showed that PP2A activators synergise with clinically relevant tyrosine kinase inhibitors to inhibit colony formation and proliferation. Therefore, the work in this thesis significantly contributes to our understanding of the role of PP2A as a tumour suppressor in AML, and suggests thatPP2A activators present a novel therapeutic strategy for RTK driven leukaemias used as a monotherapy or in conjunction with targeted tyrosine kinase inhibitors.
- Subject
- PP2A; c-KIT; FLT3; Acute Myeloid Leukaemia (AML)
- Identifier
- http://hdl.handle.net/1959.13/1043096
- Identifier
- uon:14165
- Rights
- Copyright 2014 Amanda Melanie Smith
- Language
- eng
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| Thumbnail | File | Description | Size | Format | |||
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| View Details Download | ATTACHMENT01 | Abstract | 207 KB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Thesis | 11 MB | Adobe Acrobat PDF | View Details Download |