Characterising the effects of FLT3 inhibitor AC220 on haematopoiesis and myeloproliferative neoplasms

Samuel John Taylor

    Research output: ThesisDoctoral Thesis

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    Abstract

    Leukaemia is a life-threatening disease driven by excessive activation of growth and
    survival signalling pathways in haematopoietic progenitor and stem cells. FMS-like
    tyrosine kinase 3 (FLT3) signalling is one such pathway, with commonly found
    mutations conferring overactive signalling. FLT3 internal tandem duplication (ITD)
    mutations drive constitutive activation of the receptor and are one of the most frequent
    mutations found in acute myeloid leukaemia (AML). Furthermore, c-Cbl RING finger
    and linker domain mutations, or mixed lineage leukaemia (MLL) fusions, lead to
    enhanced wild type (WT) FLT3 signalling, and are commonly found in chronic/juvenile
    myelomonocytic leukaemia or MLL patients respectively. Prominence of FLT3
    signalling in leukaemia has led to the generation of many small molecule inhibitors
    against FLT3, however the ability of these compounds to effectively treat leukaemia has
    not been fully characterised.

    AC220, or quizartinib, is a second-generation FLT3 inhibitor that possesses the most
    favourable pharmacokinetic and pharmacodynamic properties of current FLT3
    inhibitors. AC220 is being investigated in phase II clinical trials for the treatment of
    FLT3-ITD+ AML after demonstrating success in pre-clinical and phase I studies.
    Furthermore, it has been identified that AC220 is an effective treatment for a
    myeloproliferative disease (MPD) driven by enhanced WT FLT3 signalling in a mouse
    expressing a c-Cbl RING finger domain mutation, i.e. the c-CblA/- mouse. However, the
    mechanistic effect AC220 exerts upon the haematopoietic progenitor cells responsible
    for disease development remains unknown. The predominant aim of this thesis was to
    gain insight into the consequences of AC220 exposure upon haematopoiesis in
    leukaemic and WT mouse models.

    The dosing of c-CblA/- mice with AC220 caused a rapid reduction of the markedly
    expanded multipotent progenitor (MPP) population. This occurred as a result of a
    transient induction of quiescence in the MPPs, an effect AC220 instigated over the first
    four days of dosing. Interestingly, after 8 days of dosing the quiescence was reversed
    and the remaining MPPs became highly proliferative due to an induction of FLT3
    ligand (FL). The FL-induced proliferative response prevented further loss of MPPs, and
    likely prevented haematopoietic failure. The quiescence induced by AC220 in c-CblA/-MPPs
    was also evident in WT mice, implicating WT FLT3 signalling as a key driver of
    MPP proliferation.

    The transient quiescence induced by AC220 was employed to protect WT MPPs from
    cytotoxic killing by 5-fluorouracil (5-FU). A single priming dose of AC220, 6-12 hours
    prior to a 5-FU injection, was sufficient to increase the survival of MPPs 10-fold.
    Protection of the MPPs by AC220 enabled the haematopoietic system to recover from
    multiple rounds of 5-FU injections, thereby preventing the development of lethal
    myelosuppression. Thus, the findings from this thesis have identified a potential way to
    protect cancer patients from chemotherapy-induced myelosuppression, one of the most
    severe side effects of chemotherapy.

    AC220 dosing was also examined in a mutant mouse model of FLT3-ITD MPD and,
    surprisingly, it was found that the MPP population was not induced into quiescence.
    This was later explained by an AC220 resistance-conferring mutation in FLT3 in this
    mouse. Yet, despite the presence of this mutation, AC220 exerted beneficial effects
    upon the WBC counts of FLT3-ITD mice, and this was attributed to the secondary
    inhibitory actions of AC220 upon c-Kit. Furthermore, it was found that a xenograft
    mouse model of human MLL-AF9 driven leukaemia could not be effectively treated
    with AC220, providing some clarification that FLT3 signalling may not be a major
    driver of MLL.

    As demonstrated in initial studies with WT and CblA/- mice, a high level of FL is a
    significant barrier to effective inhibition with FLT3 inhibitors such as AC220. Further
    examination of this phenomenon was carried out using FLKO (FLT3 Ligand Knock
    Out):WT chimaeric mice and it was identified that a non-haematopoietic compartment
    is the predominant producer of AC220-induced FL. Future research into the specific
    signalling pathways that promote FL production is warranted as this could ultimately
    identify targets to enhance the effectiveness of FLT3 inhibitors.

    Finally, in another avenue of investigation, the role of c-Cbl in regulating FLT3-ITD
    was studied by crossing c-CblA/- and FLT3-ITD mice. c-Cbl has been well characterised
    as an E3 ubiquitin ligase that downregulates WT FLT3, but its role as a negative
    regulator of FLT3-ITD is unclear. The double mutant mice developed a rapidly fatal
    myeloid leukaemia, suggesting strong co-operation between the two mutations.
    However, there was no evidence for direct regulation of c-Cbl upon FLT3-ITD from
    protein or signalling assays. In fact it was determined that FLT3-ITD signalling
    independently cooperated with c-Cbl RING finger mutant-driven activation of WT
    tyrosine kinases, such as c-Kit, to generate the aggressive myeloid leukaemia. This
    study highlighted that FLT3-ITD protein can escape the negative regulation of c-Cbl.
    Additionally, these findings illuminate the importance of c-Cbl’s negative regulation of
    WT tyrosine kinase signalling in suppressing the development of myeloid leukaemia in
    a FLT3-ITD mouse model.
    Original languageEnglish
    QualificationDoctor of Philosophy
    Awarding Institution
    • The University of Western Australia
    Award date13 Jul 2016
    Publication statusUnpublished - 2015

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