Modulation of Pim Kinase Activity to Sensitize Acute Myeloid Leukemia (AML) Cells with FLT3-ITD to Chemotherapy

Abstract

Internal tandem duplication of the fms-like tyrosine kinase-3 (FLT3-ITD) receptor tyrosine kinase is present in acute myeloid leukemia (AML) cells of 30 percent of patients. While patients with FLT3-ITD usually achieve complete remission following induction chemotherapy, they have a high relapse rate and short disease-free survival. FLT3 inhibitors have had only limited and transient activity in clinical trials. The serine/threonine kinase Pim-1 is a pro-survival oncogene that is transcriptionally upregulated by FLT3-ITD. It promotes FLT3-ITD signaling in a positive feedback loop. Thus Pim-1 is an attractive target for AML with FLT3-ITD. In this study we evaluated the efficacy of Pim kinase inhibition in sensitizing AML cells with FLT3-ITD to chemotherapy drugs, including the nucleoside analog cytarabine and topoisomerase 2 inhibitors. We found that co-treatment with the pan-Pim kinase inhibitor AZD1208 or expression of a kinase-dead Pim-1 mutant sensitizes FLT3-ITD cell lines to apoptosis triggered by topoisomerase 2 inhibitor, but not by cytarabine. Pim kinase inhibition also sensitizes primary AML cells with FLT3-ITD, but not AML cells with wild-type FLT3 or remission bone marrow cells, to topoisomerase 2 inhibitors, supporting a favorable therapeutic index. Mechanistically, Pim kinase and topoisomerase 2 inhibitor co-treatment was associated with increased DNA double-strand breaks (DSBs) and increased induction of reactive oxygen species (ROS), which further induces DNA damage, causing the subsequent progressive increased apoptosis. Of note, enhanced apoptosis was abrogated by pretreatment with the ROS scavenger N-acetyl cysteine. We hypothesized that the increase in topoisomerase 2-induced DNA DSBs with Pim kinase inhibition might result at least in part from effects on DNA repair pathways. AZD1208 was found to inhibit alternative non-homologous end-joining (Alt-NHEJ) activity induced by the topoisomerase 2 inhibitor in cells with FLT3-ITD, in conjunction with a decrease in nuclear expression of key Alt-NHEJ proteins XRCC1 and DNA ligase 3. AltNHEJ is a highly error-prone DNA DSB repair pathway that is upregulated in cells with FLT3-ITD. Future work will determine the effect of decreased Alt-NHEJ activity on genomic instability in these cells. Overall, our data support testing of Pim kinase inhibitors with topoisomerase 2 inhibitors to improve treatment outcomes in AML with FLT3-ITD