Pim kinase inhibition sensitizes FLT3-ITD acute myeloid leukemia cells to topoisomerase 2 inhibitors through increased DNA damage and oxidative stress

// Kshama A. Doshi 1, 2 , Rossana Trotta 1, 3 , Karthika Natarajan 1, 2 , Feyruz V. Rassool 1, 4 , Adriana E. Tron 5 , Dennis Huszar 5,# , Danilo Perrotti 1, 2 , Maria R. Baer 1, 2, 6 1 University of Maryland Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD, USA 2 Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, USA 3 Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD, USA 4 Department of Radiation Oncology, University of Maryland School of Medicine, Baltimore, MD, USA 5 Oncology iMED, AstraZeneca, Waltham, MA, USA 6 Veterans Affairs Medical Center, Baltimore, MD, USA # Present address: Oncology Drug Discovery Unit, Takeda Pharmaceuticals International Co., Cambridge, MA, USA Correspondence to: Maria R. Baer, email: mbaer@umm.edu Keywords: acute myeloid leukemia, FLT3-ITD, Pim kinase, chemotherapy, reactive oxygen species Received: May 15, 2016     Accepted: June 09, 2016     Published: June 21, 2016 ABSTRACT Internal tandem duplication of fms-like tyrosine kinase-3 (FLT3-ITD) is frequent (30 percent) in acute myeloid leukemia (AML), and is associated with short disease-free survival following chemotherapy. The serine threonine kinase Pim-1 is a pro-survival oncogene transcriptionally upregulated by FLT3-ITD that also promotes its signaling in a positive feedback loop. Thus inhibiting Pim-1 represents an attractive approach in targeting FLT3-ITD cells. Indeed, co-treatment with the pan-Pim kinase inhibitor AZD1208 or expression of a kinase-dead Pim-1 mutant sensitized FLT3-ITD cell lines to apoptosis triggered by chemotherapy drugs including the topoisomerase 2 inhibitors daunorubicin, etoposide and mitoxantrone, but not the nucleoside analog cytarabine. AZD1208 sensitized primary AML cells with FLT3-ITD to topoisomerase 2 inhibitors, but did not sensitize AML cells with wild-type FLT3 or remission bone marrow cells, supporting a favorable therapeutic index. Mechanistically, the enhanced apoptosis observed with AZD1208 and topoisomerase 2 inhibitor combination treatment was associated with increased DNA double-strand breaks and increased levels of reactive oxygen species (ROS), and co-treatment with the ROS scavenger N-acetyl cysteine rescued FLT3-ITD cells from AZD1208 sensitization to topoisomerase 2 inhibitors. Our data support testing of Pim kinase inhibitors with topoisomerase 2 inhibitors, but not with cytarabine, to improve treatment outcomes in AML with FLT3-ITD.