Adaptation and Validation of the Plasma Inhibitory Activity (PIA) Assay to Detect Inhibition of Aurora, ABL and FLT3 Kinases by AT9283 In Children and Adolescents with Leukaemia

Abstract 1818

Non-invasive assessment of biomarker modulation is critically important when conducting early phase trials of targeted therapeutics, particularly in children. Measuring target modulation using primary leukaemic blasts is challenging, since obtaining bone marrow is invasive and invariably requires a general anaesthetic in children. Furthermore, in many cases only a limited number of circulating blasts can be detected in peripheral blood and these may be rapidly cleared upon commencement of effective therapy. For these two reasons, pharmacodynamic (PD) assays using surrogate biological material can be invaluable. The plasma inhibitory activity (PIA) assay was initially developed for FLT3 inhibitors and is currently used in clinical trials to assess ex vivo inhibition of FLT3 kinase and guide optimal dosing (Levis M et al, Blood 2006). Briefly, the assay involves incubating reference leukaemic cell lines in plasma from patients treated with FLT3 inhibitors then assessing the degree of FLT3 inhibition by western blotting. Inhibition of FLT3 in leukemia cell lines has been shown to correlate with inhibition in primary leukaemic blasts. Aurora kinase inhibitors are emerging as promising new agents with activity in leukaemia (Moore AS et al, Leukemia 2010). AT9283 is a novel Aurora kinase inhibitor with activity against the secondary kinase targets FLT3 and ABL (Howard S et al, J Med Chem 2009). We therefore hypothesised that the PIA assay would be applicable as an ex vivo PD assay to simultaneously detect inhibition of these target kinases in leukaemic cell lines: Aurora with FLT3 and Aurora with ABL. Furthermore, we hypothesised that the assay could be adapted to paediatric patients, where limited volumes of blood are available for PD studies. Here we report the validation of the PIA assay for use with AT9283 using leukaemic cell lines incubated in human plasma spiked with clinically relevant concentrations of AT9283 known to inhibit Aurora kinase in primary leukaemic blasts. The PIA assay proved to be a robust means of semi-quantitatively detecting concentration-dependent inhibition of Aurora kinase in the FLT3-ITD positive AML cell line MOLM-13 and the CML cell line K-562. The PIA assay was also effective at qualitatively detecting the inhibition of FLT3 signaling in MOLM-13 cells and ABL signaling in K-562 cells. Of importance to paediatric patients, the assay was successfully performed using 0.5 mL of plasma, half the volume described in the original PIA assay for FLT3 inhibitors. It will be used to assess target kinase modulation in a phase I trial of AT9283 in children and adolescents with relapsed and refractory acute leukaemia (EudraCT No. 2009–016952-36). In conclusion, the PIA assay is applicable not only to FLT3 inhibitors, but also Aurora kinase inhibitors and potentially, other multi-kinase inhibitors. By simultaneously detecting multiple kinase inhibition, the PIA assay may help delineate important mechanisms of action for novel anti-leukaemic drugs.