The pim kinase inhibitor AZD1208 enhances apoptosis induction by clinically active FLT3 inhibitors in FLT3-ITD acute myeloid leukemia cells and through synergistic downregulation of Mcl-1 and of Bcl-xL. 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 restorative index. Mechanistically, the enhanced apoptosis observed with AZD1208 and topoisomerase 2 inhibitor combination treatment NU 1025 was associated with improved DNA double-strand breaks and improved levels of reactive oxygen varieties (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 screening of NU 1025 Pim kinase inhibitors with topoisomerase 2 inhibitors, but not with cytarabine, to improve treatment results in AML with FLT3-ITD. into the cytoplasm (Number ?(Number5A,5A, Supplementary Number S4A, S4B), relative to topoisomerase 2 VASP inhibitors alone, consistent with increased intrinsic cell death signaling. Additionally co-treatment with AZD1208 and DNR, compared to DNR only, caused more pronounced cleavage of caspase 3 and its substrate PARP (Number 5A, 5B). Improved caspase 3 cleavage was also seen in Ba/F3-ITD cells co-treated with AZD1208 and VP-16 or MXR (Supplementary Number S4A, S4B). Moreover, enhanced caspase 3 cleavage was clogged and apoptosis was decreased by co-incubation with the pan-caspase NU 1025 inhibitor Z-VAD FMK (P<0.0001) (Supplementary Number S4C), highlighting the part of caspase activation in enhanced apoptosis induction by AZD1208 and topoisomerase 2 inhibitor co-treatment. NU 1025 Open in a separate window Number 5 Pim kinase inhibitor and topoisomerase 2 inhibitor co-treatment raises intrinsic cell death signaling in FLT3-ITD cellsA. AZD1208 and topoisomerase 2 inhibitor co-treatment induces loss of mitochondrial membrane potential (MMP), increase in cytoplasmic cytochrome and cleavage of caspase 3. Ba/F3-ITD cells cultured with AZD1208 and/or DNR were collected at 48 hours. To measure MMP, cells were incubated with JC-1 dye and median reddish fluorescence was measured. To measure cytoplasmic cytochrome and caspase 3 cleavage, cells were permeabilized, fixed, clogged and incubated with FITC-labeled anti-Cytochrome and FITC-labeled anti-Active Caspase 3, respectively. Means S.E.M. of triplicate experiments are demonstrated. B. AZD1208 and topoisomerase 2 inhibitor co-treatment raises PARP cleavage. Ba/F3-ITD cells were cultured with AZD1208 and/or DNR. Total cell lysates were resolved by SDS-PAGE and immunoblotted with PARP and GAPDH main antibodies. Representative immunoblots are demonstrated. C. Co-treatment with AZD1208 does not increase intrinsic cell death signaling induced by AraC. Ba/F3-ITD cells were cultured with AZD1208 and/or AraC. MMP, cytoplasmic cytochrome and caspase 3 cleavage were measured as explained above. Means S.E.M. of triplicate experiments are shown. launch or caspase 3 cleavage in Ba/F3-ITD cells, and actually modestly safeguarded from AraC-induced loss of MMP (P<0.0001) (Number ?(Figure5C)5C) and decreased cytochrome release (P<0.01) (Number ?(Figure5C)5C) and caspase 3 cleavage (P<0.01) (Number ?(Number5C5C). Pim kinase inhibitor enhances induction of DNA damage and ROS generation by topoisomerase 2 inhibitors, but not by AraC, in cells with FLT3-ITD Topoisomerase 2 inhibitors stabilize the topoisomerase 2 enzyme during DNA replication, therefore causing collapse of the replication fork, which results in DNA double-strand breaks (DSBs) and subsequent cell death [27, 28]. Phosphorylated histone H2AX (-H2AX), a marker for DNA DSBs [29, 30], improved more than two-fold within 8 hours of concurrent treatment of Ba/F3-ITD cells with AZD1208 and topoisomerase 2 inhibitors, relative to topoisomerase 2 inhibitors only, with subsequent sustained increase (Number ?(Number6A,6A, Supplementary Number S5A, S5B). DNA damage induces oxidative stress, which leads to further DNA damage, developing a positive opinions loop that triggers cell death [31, 32]. AZD1208 and topoisomerase 2 inhibitor combination treatment caused minimal induction of ROS in Ba/F3-ITD cells up to 24 hours, followed by a two-fold increase at later time points, relative to treatment with topoisomerase 2 inhibitors only (Number ?(Number6B,6B, Supplementary Number S6A). Pretreatment of Ba/F3-ITD cells with the ROS scavenger NAC reduced ROS induction (Supplementary Number S6B), as expected, and decreased induction of -H2AX manifestation by the combination treatment (Number ?(Number6C,6C, remaining). Moreover, apoptosis was markedly attenuated when Ba/F3-ITD cells were treated with NAC before AZD1208 and topoisomerase 2 inhibitor combination treatment (P<0.001) (Number ?(Number6C,6C, right, Supplementary Number S6C). Finally, the lack of potentiation of AraC-induced apoptosis by Pim kinase inhibition displays reduced AraC-mediated DNA damage (Number ?(Figure7A)7A) and.