Abstract 5332: The Pim kinase inhibitor AZD1208 sensitizes acute myeloid leukemia cells with fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) to cytotoxic effects of chemotherapy drugs
Authors: Doshi Kshama A. , Natarajan Karthika , Wolfson Benjamin , Huszar Dennis , Baer Maria R. 
Published: August 2015

Abstract
Purpose: Internal tandem duplication (ITD) in the juxtamembrane domain of the fms-like tyrosine kinase 3 (FLT3) receptor is present in leukemia cells of approximately 30% of acute myeloid leukemia (AML) patients. These patients have a high remission rate, but short relapse-free and overall survival. Pim-1 kinase, a pro-survival oncogene, is transcriptionally upregulated downstream of FLT3-ITD and promotes FLT3-ITD signaling via a positive feedback loop. We proposed that Pim kinase inhibitors would enhance the cytotoxic effects of chemotherapy drugs in AML cell lines and patient samples with FLT3-ITD.
Methods: AZD1208, a pan-Pim (Pim-1, Pim-2 and Pim-3) kinase inhibitor, was kindly provided by AstraZeneca. FLT3-ITD cell lines included MV4-11, MOLM-14 and Ba/F3-ITD. The topoisomerase 1 and 2 inhibitors daunorubicin, mitoxantrone, etoposide and topotecan and the nucleoside analog cytarabine were studied at their IC50 concentrations for each cell line. Apoptosis was measured by flow cytometric analysis of Annexin V/propidium iodide (PI) staining, percent cells in sub-G1 cell cycle phase using PI staining and mitochondrial membrane potential (MMP) using JC-1 dye, as well as flow cytometric and western blot measurements of cleaved poly (ADP-ribose) polymerase (PARP) and caspase-3.
Results: Co-treatment with 1μM AZD1208 and daunorubicin induced significantly more apoptosis than daunorubicin or AZD1208 alone (e.g., 65% vs. 25% and 7% in Ba/F3-ITD) in FLT3-ITD cell lines. At least two-fold increase in apoptosis was also seen when AZD1208 was combined with mitoxantrone, etoposide or topotecan, compared with each drug alone, but AZD1208 did not increase apoptosis induced by cytarabine. Chemosensitization of FLT3-ITD cells with the combination treatment was confirmed by a higher percentage of sub-G1 cells (e.g., 33% for AZD1208 and daunorubicin combination vs. 17% for daunorubicin alone in Ba/F3-ITD), more rapid loss of MMP and increased time-dependent cleavage of PARP and caspase-3. Apoptosis was significantly (P&lt;0.0001) rescued by co-treatment with the pan-caspase inhibitor z-VAD. Cell lines with wild-type FLT3 were less sensitive to combined AZD1208 and chemotherapy-induced apoptosis. Finally, increased cytotoxicity of daunorubicin in the presence of AZD1208 was also seen in FLT3-ITD AML patient marrow samples, but not remission marrow samples.
Conclusion: Our work supports clinical applicability of combining a Pim kinase inhibitor with chemotherapy to treat AML with FLT3-ITD. Ongoing work is directed toward determining the molecular mechanisms of sensitization of FLT3-ITD cells to chemotherapy by Pim kinase inhibition.
Citation Format: Kshama A. Doshi, Karthika Natarajan, Benjamin Wolfson, Dennis Huszar, Maria R. Baer. The Pim kinase inhibitor AZD1208 sensitizes acute myeloid leukemia cells with fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) to cytotoxic effects of chemotherapy drugs. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5332. doi:10.1158/1538-7445.AM2015-5332