Abstract 1050: Mechanisms of action of Pim kinase inhibitor, AZD1208, in acute myeloid leukemia cells.
Authors: Chen Lisa S. , Cortes Jorge E. , Gandhi Varsha 
Published: August 2014

Pim kinase proteins (Pim-1, -2 and -3) are a family of conserved proto-oncogenes that are upregulated in leukemias and lymphomas and are involved with leukemogenesis. Pim kinase substrates include transcription, translation, cell cycle, and survival proteins. Increased Pim expression in acute myeloid leukemia (AML), especially in FLT3-ITD activating mutation, provides a rationale to evaluate Pim kinase inhibitors in AML. AZD1208 is a small molecule pan-Pim kinase inhibitor [IC50: Pim-1 (0.4 nM), Pim-2 (5.0 nM) and Pim-3 (1.9 nM)]. AZD1208 treatment resulted in growth inhibition in AML cell lines including FLT3-WT (OCI-AML-3, KG-1a, MOLM-16) and FLT3-ITD mutated (MOLM-13, MV-4-11), with greater sensitivity measured in MOLM-16, followed by KG-1a and MV-4-11. The effects of AZD1208 on cell survival were investigated and there was limited apoptosis induction (&lt;10% increase) in the AML cell lines evaluated as measured by flow cytometry and annexin-V/propidium-iodide staining after 24 h with up to 3 μM AZD1208. This was consistent with a lack of decline in Mcl-1 protein levels, suggesting that growth inhibition is not through apoptosis induction. Preliminary data also indicated that there was limited change in cell cycle profile in OCI-AML-3 or MV-4-11 cells treated with AZD1208 for up to 72 h, suggesting that growth inhibition is not attributed to cell cycle arrest.

To elucidate the mechanism of AZD1208, Pim kinase target protein phosphorylation was evaluated. With 3 μM AZD1208 for 24 h, there was a decrease in Bad (Ser112) phosphorylation relative to total Bad only in KG-1a and MOLM-16 cells. Similarly, there was significant decline in histone H3 (Ser10) phosphorylation only in MOLM-16. However, several mTOR signaling pathway proteins were modulated by AZD1208. Pim-1 target PRAS40 (Thr246) phosphorylation decreased in KG-1a, MOLM-13, MV-4-11 and MOLM-16 within 6 h of exposure to 3 μM AZD1208. Similarly, Pim-2 target 4E-BP1 (Ser65) phosphorylation decreased in all five cell lines by 24 h. Phosphorylation of downstream S6 (Ser235/236) was dramatically reduced in KG-1a and MOLM-16, and also declined in MV-4-11. Growth inhibition mediated by AZD1208 is similar in FLT3-WT (KG-1a) and FLT3-ITD (MV-4-11), however there is a varied response in Bad and S6 phosphorylation. These results indicate that although growth inhibition induced by AZD1208 is similar in cells with disparate FLT3 mutation status, the molecular events are divergent and may uncover signaling differences between Pim and FLT3-WT and FLT3-ITD. Taken together, Pim kinase inhibition by AZD1208 may be a valuable strategy to inhibit AML cell proliferation even in hard-to-treat AML with FLT3 activating mutations, and these investigations will be used to identify biomarkers during clinical trial that may be predictive of response in patients. There is a multi-center Phase I clinical trial with AZD1208 in AML currently underway and investigations are ongoing.

Citation Format: Lisa S. Chen, Jorge E. Cortes, Varsha Gandhi. Mechanisms of action of Pim kinase inhibitor, AZD1208, in acute myeloid leukemia cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1050. doi:10.1158/1538-7445.AM2013-1050