The Effect of ABC Transporters and FLT3 Status on the Activity of the Aurora Kinase B Inhibitor AZD1152 in AML Cells.

The aurora kinases are essential for mitotic progression. Both Aurora A and B are over-expressed in a wide variety of human haematological and epithelial malignancies. A number of Aurora kinase inhibitors have been described that show anti-tumour activity in vivo, with results suggesting that their cellular effects are largely due to the inhibition of Aurora B. Aurora B is required for histone H3 phosphorylation, chromosome segregation and cytokinesis. AZD1152 is an aurora kinase inhibitor with potent activity against Aurora B. The compound is a phosphate prodrug that is converted rapidly in vivo to the active moiety AZD1152-HQPA. Acute myeloid leukaemia (AML) is a heterogeneous malignancy that affects bone marrow haematopoietic cells of the myeloid lineage. The ABC transporter P-glycoprotein (Pgp) is a known poor prognostic factor in the treatment of AML. Breast cancer resistance protein (BCRP) has been implicated in the resistance to chemotherapy in AML. AZD1152-HQPA activity was studied in 6 myeloid cell lines: KG1a (Pgp+, BCRP−), U937 (−,−), MV4-11(−,−), MOLM-13 (−,−), OCI-AML3 (−,−) and BCRP transfected OCI-AML6.2 (−, +) at concentrations of 0–1000nM. The Pgp and BCRP status of cell lines was confirmed with R123 and BODIPY-prazocin flow cytometric accumulation assays. Flow cytometry was used to determine 24hr inhibition of phosphorylation of histone H3 (phH3) as a biomarker for AZD1152-HQPA activity as well as 72hr cell number and viability using an in-house flow cytometric method. Both the Pgp positive KG1a cells and the OCI-AML6.2 cells showed decreased sensitivity to AZD1152-HQPA compared with transporter-negative cell lines. This was noted at the level of the biomarker phH3 as well as growth inhibition. Interestingly, the most sensitive lines were the FLT3-ITD expressing MV4-11 and MOLM-13 cells, with cell viability IC50s below 10nM and complete down-regulation of phH3 at 30nM. FLT3 mutations are found in approximately 30% of AML patients, either FLT3-ITD (24%) or FLT-3 activation loop mutations (7%), making FLT3 mutation one of the most commonly mutated genes in AML. ITD mutation of FLT3 induces activating phosphorylation of the receptor in the absence of ligand. Using immunoprecipitation we were able to show a ≥40% decrease in phosphorylated FLT3 (pFLT3) expression in the ITD cell lines after 1hr treatment with 300nM AZD1152-HQPA and a similar decrease in phosphorylation of the downstream transcription factor STAT5 (measured by flow cytometry). AZD1152-HQPA activity was studied in 14 primary AML samples after 48hr pre-incubation with a cytokine cocktail to induce cycling. Pgp status of the samples was confirmed by cytometry using R123 and MRK16 antibody. BCRP and FLT3 status of the samples was determined using RT-PCR. 5 samples were positive for Pgp, 4 of which were also positive for BCRP, and 5 samples had a FLT3-ITD mutation. 20% inhibition of phH3 was achieved in all 14 samples at concentrations ≤130nM AZD1152-HQPA after 1hr. 20% cell death was achieved in 11 out of 12 samples at ≤300nM AZD1152-HQPA, including 3 out of 4 Pgp+ and 2 out of 3 BCRP+ samples. There was a statistical trend towards increased sensitivity for the ITD samples (IC20 ≤15nM; p=0.09). 40% inhibition of pSTAT5 was achieved in 2 ITD samples after 1hr treatment with 100 nM AZD1152-HQPA. In conclusion, the decreased response to AZD1152-HQPA in Pgp+ and BCRP+ cell lines is not characteristic of patient samples. AZD1152-HQPA directly targets pFLT3 in FLT3ITD cells along with its downstream target pSTAT5. AZD1152 is currently being tested in phase I clinical trials.