Tinsley, SP; (2014) To establish the role of mutations in c-KIT tyrosine kinase in the pathogenesis and therapy of core-binding factor-related acute myeloid leukaemia (AML). Doctoral thesis , UCL (University College London).

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Abstract

Haematopoiesis is controlled by complex signal transduction pathways and transcription regulators which may become dysregulated in acute myeloid leukaemia (AML). Activating mutations in FLT3 and c-KIT receptor tyrosine kinases (RTKs) are commonly found in AML and can impact on prognosis. Different types of FLT3 mutations are known to have distinct biological activities and prognostic implications. The presence of c-KIT mutations has been shown to increase the risk of relapse, but there has been no direct comparison of the biological activity of AML specific c-KIT mutations occurring in different receptor domains. In addition to prognostic information, RTKs are attractive potential targets for therapy using small molecule inhibitors. To evaluate their biological activity and response to targeted therapies, human UT-7 cells were transduced with wild-type and mutant c-KIT isoforms - c-KIT-Δ417-419>Y (extracellular domain [ECD]), c-KIT-L576P (juxtamembrane domain [JMD]), c-KIT-D816V and c-KIT-N822K (both in the activating loop domain [ALD). There were intrinsic differences in signal strength between the mutants examined - only c-KIT-Δ417-419>Y and c-KIT-D816V expressing cells had detectable constitutive c-KIT activation and showed ligand-independent growth. The response of transduced UT-7 cells to c-KIT inhibitors was assessed by treating the cells with dasatinib and masitinib. Cells expressing ALD c-KIT mutations were more resistant to dasatinib or masitinib-mediated cell killing in comparison to cells expressing c-KIT mutations in the ECD and JMD. Western blotting revealed that although c-KIT phosphorylation was potently inhibited, there was residual mTOR and/or PI3K/AKT activation in these cells. The resistance to dasatinib observed in c-KIT-D816V or c-KIT-N822K expressing cells could be overcome by co-blockade of c-KIT and PI3K/mTOR and blockade of c-KIT and PI3K/mTOR was synergistic in all c-KIT mutant cell lines at inducing cell death. Blockade of FLT3 and PI3K/mTOR in FLT3-ITD AML cell lines also showed similar results. During the screening of AML cell lines for c-KIT and FLT3 mutations a novel FLT3-T820N point mutation was identified in the ME-1 cell line. Expression of FLT3-T820N in 32D cells constitutively activated FLT3 and conferred ligand-independent growth. 32D FLT3-T820N cells were most sensitive to the FLT3 inhibitor AC220 with regard to growth inhibition, cell killing and decreased phosphorylation of FLT3 compared to FLT3-WT and FLT3-D835Y expressing cells. This work highlights the differences in biological outcomes and TK inhibitor-sensitivity between different RTK mutants found in AML and shows that simultaneous blockade of RTKs and PI3K/mTOR may provide a novel therapeutic approach for specific AML subtypes.

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