FLT3 Signalling Pathways in Acute Myeloid Leukaemia

Abstract

FMS-like tyrosine kinase 3 (FLT3) is a class III receptor tyrosine kinase. Mutations affecting the FLT3 gene are frequent in acute myeloid leukaemia (AML) and are detected in up to 35% of patients, where they are associated with shorter remission rates and higher relapse risk. FLT3 is therefore an important potential target for therapy of AML. However, to date, inhibitors of FLT3 tyrosine kinase have had limited clinical success. This thesis employs cell line models of AML assessed by proteomic analysis, aiming to address whether proteins associated with FLT3-response can be identified and could form part of novel strategies to improve outcome. Two cell lines: MV4-11 (FLT3/ITD) and OCI-AML3 (wtFLT3) were assessed for their suitability as models to test FLT3 ligand (FL) or FLT3-inhibitor effect. Each was shown to express functional FLT3 receptor and to upregulate pERK signalling in response to FL, which was inhibited in each case by FLT3 inhibitor drugs. However, the FLT3/ITD cell line MV4-11, but not OCI-AML3, also demonstrated high baseline signal activation of pERK and in cell biological testing Quizartinib induced apoptosis and abolished proliferation only in FLT3/ITD MV4-11 cells. The FLT3 inhibitor Quizartinib was selected for detailed study, based on literature evaluation and on dose-dependent inhibition of pERK in each cell line. A proteomic approach (Sequential Window Acquisition of all theoretical fragment-ion spectra mass spectrometry (SWATH-MS)) was then used to identify whether protein expression differed between the sensitive (MV4-11) and resistant cell (OCI-AML3) lines at baseline or in conditions of stimulation or inhibition. These analyses showed that MV4-11 and OCI-AML3 lines differed in their response to Quizartinib, but that the most marked changes required the additional presence of FL. Bioinformatic analysis of responses in the different conditions demonstrated that FLT3 pathways predominantly involved the processes of cell growth, apoptosis or replication (consistent with the findings in section 3.1). The changes affecting those processes were much more marked in MV4-11 cells, and the bioinformatic study of the proteins identified candidate molecules for therapeutic targeting such as BCL2 and ROCK1 pathway. Although these findings should be regarded as preliminary and requiring confirmation in larger test sets, this thesis shows the potential value of this approach to identify additional target proteins for therapy in patients with FLT3/ITD mutated AML.

Date of Award	31 Dec 2020
Original language	English
Awarding Institution	The University of Manchester
Supervisor	John Burthem (Supervisor) & Karen Rees-Unwin (Supervisor)