The Pim Kinase Inhibitor AZD1208 Enhances Topoisomerase-2 Inhibitor Induction of Apoptosis of Acute Myeloid Leukemia Cells with FLT3-ITD through Increased Oxidative Stress
Authors: Doshi Kshama A. , Natarajan Karthika , Rassool Feyruz V. , Huszar Dennis , Baer Maria R. 
Published: October 2019

The receptor tyrosine kinase fms-like tyrosine kinase-3 (FLT3) is expressed on acute myeloid leukemia (AML) cells in most patients, and is mutated most commonly by internal tandem duplication (ITD), in approximately 30% of patients, resulting in constitutive activation and growth signaling. Initial treatment for AML consists of combination chemotherapy including cytarabine (AraC) and a topoisomerase-2 (TOP-2) inhibitor, most commonly daunorubicin (DNR). Other TOP-2 inhibitors, including mitoxantrone (Mitox) and etoposide (VP-16) are also active in AML. AML patients with FLT3-ITD achieve remission, but have high relapse rates and short disease-free survival. FLT3 inhibitors are in clinical trials, but so far with limited and transient activity. Hence new therapeutic approaches targeting additional signaling molecules are needed.
Pim-1 kinase, a pro-survival oncogene, is transcriptionally upregulated downstream of FLT3-ITD and promotes its signaling in a positive feedback loop. We have shown that the Pim kinase inhibitor AZD1208 enhances apoptosis induction of FLT3-ITD cells by FLT3 inhibitors, both in vitro and in vivo. We hypothesized that Pim-1 kinase inhibition might also enhance apoptosis induction by chemotherapy drugs.
Ba/F3 cells transfected with FLT3-ITD (Ba/F3-ITD) were treated with DNR, Mitox, VP-16 or AraC at their approximate IC50 concentrations with and without 1µM AZD1208, and viable cells were counted. While growth of Ba/F3-ITD was reduced by DNR, Mitox, VP-16 or AraC, it was abrogated by co-treatment with AZD1208 and DNR, Mitox or VP-16, but not AraC. Ba/F3 cells transfected with wild type (WT) FLT3 (Ba/F3-WT) were less sensitive to this effect. To determine whether the effect on growth was cytostatic or cytotoxic, we studied cell cycle by flow cytometric analysis of propidium iodide (PI) staining. A significant increase in Ba/F3-ITD cells in sub-G1 phase was seen following treatment with AZD1208 and TOP-2 inhibitors, compared to TOP-2 inhibitors alone, consistent with enhanced apoptosis. Significant (P<0.0001) increase in apoptosis in multiple FLT3-ITD cell lines, including transfected Ba/F3-ITD and 32D-ITD cells and human AML cells MV4-11 and MOLM-14, co-treated with AZD1208 and TOP-2 inhibitors was confirmed using Annexin V/PI staining. Minimal or no effect was seen with combination treatment in Ba/F3-WT or 32D-WT cells. Further, treatment of Ba/F3-ITD cells with AZD1208 and TOP-2 inhibitors, in relation to TOP-2 inhibitors alone, accelerated loss of mitochondrial membrane potential (MMP) and increased cytochrome c release, caspase-3 activation and PARP cleavage. Enhanced induction of apoptosis (P<0.001) was also seen in FLT3-ITD AML patient samples co-treated with AZD1208 and TOP-2 inhibitors, in relation to TOP-2 inhibitors alone, but was not seen in FLT3-WT AML patient samples, nor in AML remission bone marrow samples, with the latter observation supporting a favorable therapeutic index. Consistent with the effects on cell growth, co-treatment with AZD1208 did not sensitize Ba/F3-ITD cells to apoptosis induction by AraC, and AZD1208 actually modestly decreased AraC-induced apoptosis in Ba/F3-ITD (P<0.0001) and 32D-ITD (P<0.01) cells. Additionally co-treatment with AZD1208 and AraC decreased AraC-induced loss of MMP, release of cytochrome c and caspase-3 activation in Ba/F3-ITD cells (P<0.001).
Mechanistically, when compared to TOP-2 inhibitors alone, co-treatment with AZD1208 and TOP-2 inhibitors increased oxidative stress in Ba/F3-ITD cells, as evidenced by more than two-fold increased generation of reactive oxygen species (ROS). Further, treatment with the ROS scavenger N-acetyl cysteine markedly attenuated the increase in apoptosis produced by AZD1208 and TOP-2 inhibitors, consistent with a causative role of ROS production in apoptosis induction, through increased DNA damage. Finally, consistent with the lack of increase in apoptosis, co-treatment with AZD1208 and AraC did not increase ROS generation.
We conclude that Pim kinase inhibition enhances induction of apoptosis of AML cells with FLT3-ITD by TOP-2 inhibitors, but not AraC, through increased oxidative stress. The selective effect in FLT3-ITD cells may be due to high baseline ROS levels and/or Pim-1 kinase overexpression. Our work supports potential clinical applicability of combining a Pim kinase inhibitor with TOP-2 inhibitors, but not AraC, to treat AML with FLT3-ITD.

Disclosures
Huszar: Astra Zeneca: Employment.