Abstract 3866: The clinically applicable pan-Pim kinase inhibitor PIM447 sensitizes acute myeloid leukemia cells with FLT3-ITD to induction of apoptosis by FLT3 inhibitors and by topoisomerase 2 inhibitors
Authors: Doshi Kshama A. , Baldwin Patrick R. , Kapoor Shivani , Baer Maria R. 
Published: July 2016

Abstract
Introduction:
Internal tandem duplication of the fms-like tyrosine kinase-3 receptor (FLT3-ITD) is present in acute myeloid leukemia (AML) cells in 30% of patients and these patients have short disease-free survival following chemotherapy. FLT3 inhibitors are clinically active, but their activity is limited and transient. The oncogenic serine/threonine kinase Pim-1 is transcriptionally upregulated downstream of FLT3-ITD, and promotes FLT3 signaling in a positive feedback loop in FLT3-ITD cells. We have previously demonstrated that inhibiting Pim kinase sensitizes AML cell lines and primary AML patient blasts with FLT3-ITD to induction of apoptosis by FLT3 inhibitors and by topoisomerase 2 inhibitor chemotherapy drugs. Here we studied the effects of the pan-Pim kinase inhibitor PIM447 (formerly LGH447; Novartis Pharmaceuticals), currently in clinical trials, on response of FLT3-ITD-expressing cell lines and AML patient samples to FLT3 inhibitors and to topoisomerase 2 inhibitors, with the ultimate goal of developing clinically applicable combination regimens.
Methods:
Cell lines and AML patient samples with FLT3-ITD were cultured with FLT3 inhibitors or topoisomerase 2 inhibitors at clinically applicable concentrations and PIM447 at a range of concentrations. Apoptosis was measured by Annexin V labeling and PARP cleavage. Mcl-1 expression was measured by immunoblotting. Reactive oxygen species (ROS) were measured with the redox-sensitive dye CM-H2DCFDA and DNA double-strand breaks (DSBs) by immunoblotting for γH2AX.
Results:
Transfected Ba/F3-ITD and 32D/ITD cells, MV4-11 and MOLM-14 human AML cells and primary AML patient blasts, all expressing FLT3-ITD, were treated with FLT3 inhibitors, including 100 nM midostaurin and 1 nM quizartinib, with 0, 10, 100 and 500 nM PIM447. Co-treatment with PIM447 produced a concentration-dependent increase in apoptosis induced by each FLT3 inhibitor (p&lt;0.001). FLT3-ITD-expressing cells were also treated with the topoisomerase 2 inhibitors daunorubicin and etoposide at IC50 concentrations, with 0, 10, 100 and 500 nM PIM447. Co-treatment with PIM447 also produced a concentration-dependent increase in apoptosis induced by each topoisomerase 2 inhibitor (p&lt;0.001). Increased apoptosis induced by PIM447 with FLT3 inhibitors was associated with Mcl-1 downregulation, while increased apoptosis induced by PIM447 with topoisomerase 2 inhibitors was associated with increased generation of ROS and increased DNA DSBs.
Conclusions:
The clinically applicable pan-Pim kinase inhibitor PIM447 sensitizes AML cells with FLT3-ITD to induction of apoptosis by FLT3 inhibitors and by topoisomerase 2 inhibitors. Our data support in vivo testing of combination regimens and design of clinical trials aimed at improving outcomes for patients with AML with FLT3-ITD.
Citation Format: Kshama A. Doshi, Patrick R. Baldwin, Shivani Kapoor, Maria R. Baer. The clinically applicable pan-Pim kinase inhibitor PIM447 sensitizes acute myeloid leukemia cells with FLT3-ITD to induction of apoptosis by FLT3 inhibitors and by topoisomerase 2 inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3866.