Pim kinase inhibitor co-treatment decreases alternative non-homologous end-joining DNA repair and genomic instability induced by topoisomerase 2 inhibitors in cells with FLT3 internal tandem duplication.

Shivani Kapoor,Mario Scarpa,Prerna Singh,Julie C Dunning Hotopp,Moaath K Mustafa Ali,Aditi Chatterjee,Kshama A Doshi,Eric S Tvedte,Jonelle K Lee,Robin E Bromley,Feyruz V Rassool,Maria R Baer,Ying S Zou

doi:10.18632/oncotarget.28042

Abstract

Acute myeloid leukemia (AML) with fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) relapses with new chromosome abnormalities following chemotherapy, implicating genomic instability. Error-prone alternative non-homologous end-joining (Alt-NHEJ) DNA double-strand break (DSB) repair is upregulated in FLT3-ITD-expresssing cells, driven by c-Myc. The serine/threonine kinase Pim-1 is upregulated downstream of FLT3-ITD, and inhibiting Pim increases topoisomerase 2 (TOP2) inhibitor chemotherapy drug induction of DNA DSBs and apoptosis. We hypothesized that Pim inhibition increases DNA DSBs by downregulating Alt-NHEJ, also decreasing genomic instability. Alt-NHEJ activity, measured with a green fluorescent reporter construct, increased in FLT3-ITD-transfected Ba/F3-ITD cells treated with TOP2 inhibitors, and this increase was abrogated by Pim kinase inhibitor AZD1208 co-treatment. TOP2 inhibitor and AZD1208 co-treatment downregulated cellular and nuclear expression of c-Myc and Alt-NHEJ repair pathway proteins DNA polymerase θ, DNA ligase 3 and XRCC1 in FLT3-ITD cell lines and AML patient blasts. ALT-NHEJ protein downregulation was preceded by c-Myc downregulation, inhibited by c-Myc overexpression and induced by c-Myc knockdown or inhibition. TOP2 inhibitor treatment increased chromosome breaks in metaphase spreads in FLT3-ITD-expressing cells, and AZD1208 co-treatment abrogated these increases. Thus Pim kinase inhibitor co-treatment both enhances TOP2 inhibitor cytotoxicity and decreases TOP2 inhibitor-induced genomic instability in cells with FLT3-ITD.

Highlights

Internal tandem duplication (ITD) of the fms-like tyrosine like kinase 3 (FLT3) receptor tyrosine kinase is present in acute myeloid leukemia (AML) cells of 30 percent of patients [1], resulting in constitutive and aberrant FLT3 signaling [2]
Ba/F3-ITD cells, transfected with human FLT3-ITD, with stably integrated double-strand break (DSB) repair reporters DR-GFP, EJ2GFP or EJ5-GFP transduced with lentivirus expressing mCherry-tagged I-SceI (Supplementary Figure 1A) were treated with the topoisomerase 2 inhibitor daunorubicin and/or the pan-Pim kinase inhibitor AZD1208, or DMSO control, and harvested at 24 and 36 hours
New structural chromosome abnormalities are frequently present at relapse of AML with FLT3-ITD [4], implicating genomic instability as a significant driver in the genesis of relapse [4, 19]

Summary

Introduction

Internal tandem duplication (ITD) of the fms-like tyrosine like kinase 3 (FLT3) receptor tyrosine kinase is present in acute myeloid leukemia (AML) cells of 30 percent of patients [1], resulting in constitutive and aberrant FLT3 signaling [2]. Remission induction chemotherapy including the nucleoside analog cytarabine (AraC) and an anthracycline and/or other topoisomerase 2 inhibitor remains first-line therapy for AML [3]. Inhibition of Pim kinases is an attractive therapeutic approach in AML with FLT3-ITD. We previously showed that concurrent treatment with a Pim kinase inhibitor enhances induction of apoptosis by topoisomerase 2 inhibitor chemotherapy drugs in cells with FLT3-ITD [13]. Co-treatment with Pim kinase inhibitor increases topoisomerase 2 inhibitor induction of reactive oxygen species (ROS) and DNA double-strand breaks (DSBs) [13]. Of note, increased apoptosis occurs without changes in cell cycle distribution in residual cells, and without cell cycle arrest [13]

Methods

Results

Conclusion