Characterisation of the in vitro properties of pacritinib (SB1518) in Acute Myeloid Leukaemia

Marrin, Ceri 2015. Characterisation of the in vitro properties of pacritinib (SB1518) in Acute Myeloid Leukaemia. MPhil Thesis, Cardiff University. Item availability restricted.

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Abstract

Acute Myeloid Leukaemia remains an incurable malignancy in the majority of cases, with long term survival rates of 30-40% in those under 60 years old and less than 15% in older patients. FMS like Tyrosine kinase 3 (FLT3) is a trans-membrane receptor tyrosine kinase that is mutated in around one third of cases of AML, making FLT3 an attractive therapeutic target and paving the way for the development of a range of FLT3 Tyrosine Kinase Inhibitors (TKIs) over the last 10 years. Progress has been hampered by the limited efficacy of the majority of FLT3 TKIs in targeting the bone marrow blast population. Pacritinib is a second generation TKI with equimolar efficacy for both the FLT3 and Janus Kinase 2 (JAK2) receptors. FLT3 occupies a strategic position at the head of a complex array of downstream pathways which control transduction of signals from the extracellular environment to the nucleus. These pro-survival pathways may be enhanced on stroma leading to the creation of leukaemia niches where AML blasts can evade the effects of treatment. Cross talk between pathways such as dual activation of Signal Transducer and Activator of Transcription 5 (STAT5) by FLT3 and JAK2 has been implicated in this phenomenon and can be targeted by pacritinib. Pacritinib showed good efficacy in cell lines and in primary AML mononuclear cells with increased potency seen in cases which harboured a FLT3 ITD mutation compared to wild type FLT3. There was no correlation between surface FLT3 level, as measured by CD135 expression, and in vitro sensitivity to pacritinib. Western blotting experiments demonstrated inhibition of phosphorylated FLT3 protein in MV4-11 cell lines and rapid inhibition of phosphorylated-STAT5 in MV4-11 and primary AML cells in a dose dependent manner following pacritinib treatment. There was minimal effect on the Extracellular Regulated Kinase (ERK) pathway which is known to generate pro-survival signals in AML cells and may be upregulated on stroma. Short term resistance to pacritinib treatment was seen when primary AML cells were cultured on mouse (MS-5) stroma with supplemented medium as compared to culture in standard medium alone, potentially related to delayed apoptotic induction on stroma as seen by Annexin V Binding. Assay. Further western blotting experiments in the co-culture setting showed basal up regulation of both p-STAT5 and p-ERK in primary AML when cultured on MS-5 stroma which was not entirely inhibited by pacritinib treatment, supporting the argument that this is a possible mechanism of drug resistance. This observation led to the rationale to combine pacritinib with two different agents, which could potentially be used to eradicate residual disease within the stroma niche. A good synergistic effect was seen using the combination of pacritinib with cytarabine and with PD0325901 (a small molecule inhibitor of the ERK pathway) and this latter combination may be able to overcome stroma-induced protection of AML blasts. This justifies the on-going laboratory and clinical development of pacritinib in targeting environment mediated drug resistance in acute myeloid leukaemia.

Item Type:	Thesis (MPhil)
Date Type:	Completion
Status:	Unpublished
Schools:	Medicine
Subjects:	R Medicine > R Medicine (General)
Date of First Compliant Deposit:	30 March 2016
Last Modified:	16 May 2022 09:38
URI:	https://orca.cardiff.ac.uk/id/eprint/78043

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