Aurora Kinase Inhibition Enhances the Chemotherapy-Induced Myeloid Leukemia Cell Death through Different Mechanism According to the Chemotherapeutic Agents
Authors: Kim Soo Jeong , Jung Haeng Im , Lee Hye Won , Kim Jin Seok , Min Yoo-Hong 
Published: April 2020

Abstract
Aurora family of serine/threonine kinases plays a critical role in ensuring chromosome segregation during cell cycle, cytokinesis during mitosis, and maintaining genetic integrity in cell division. It has been shown that Aurora kinase A (AurA) and AurB were aberrantly overexpressed in a variety of human cancers including leukemia disorders, and associated with aneuploidy and poor prognosis in several cancers. Recently, it was also observed that overexpression of aurora kinase renders cancer cells to resist to chemotherapy- or radiation-induced cell death. We evaluated aurora kinase expression in leukemia cells obtained from patients with acute myeloid leukemia (AML) and analyzed its correlation with clinical parameters. Finally, we examined the effects of aurora inhibition, either with specific RNA interference (siRNA) or inhibitory molecule, on the chemotherapeutic agent-induced cell death in leukemia cell lines. Western blot analyses demonstrated that phospho(p)-AurA and p-AurB were observed in 35 (70%) and 33 (66%) of primary leukemia blasts obtained from 50 patients with de novo AML. Correlation between levels of p-AurA and p-AurB were not observed. Levels of p-AurA or p-AurB expression were significantly higher in AML blasts compared with normal bone marrow (BM) mononuclear cells (p&lt;0.001 for p-AurA and p&lt;0.001 for p-AurB, respectively). Expression level of Aur-A or Aur-B was not correlated with white blood cell counts, age, lactic dehydrogenase level, cytogenetics, complete remission rate after conventional induction chemotherapy, and disease-free survival. With silencing of AurA or AurB in U937 leukemia cell line by specific AurA and AurB siRNA transfection, the cell death was observed in 4.3 ± 0.6% and 23.2 ± 1.4%, respectively. A significant increase in the G2-M population was observed in AurA siRNA (32.8 ± 2.4%) and AurB siRNA (58.3 ± 5.3%) treatment with a concomitant decrease in the G0-G1 population compared to control cells. In vitro cell death induced by 48-hour treatment of cytosine arabinoside (AraC, 20mM) was moderate (19.3 ± 3.2%) in U937 cells. However, when AraC treatment was combined with AurA siRNA (AraC/AurA) or AurB siRNA (AraC/AurB) silencing, cell death was significantly increased to the level of 81.1 ± 7.2% (p&lt;0.005) and 77.8 ± 4.5% (p&lt;0.001), respectively. This enhancing effect was also demonstrated using aurora kinase chemical inhibitor instead of siRNA transfection. We also observed a remarkable increase in cell death extent when etoposide (50mg/ml) was combined with AurA siRNA or AurB siRNA treatment (p&lt;0.01 for Eto/AurA; p&lt;0.001 for Eto/AurB). Cell death was accompanied by disruption of mitochondrial membrane potential when AraC or etoposide was combined with AurA or AurB inhibition. However, cleavage of caspase-3, -8, -9, and PARP was not observed in the AraC/AurA or AraC/AurB treatment. In contrast, the activation of caspase cascade was observed in the Eto/AurA or Eto/AurB treatment. Both in AraC/AurA and AraC/AurB treatment, an enhanced increase in cell death was associated with multi-nucleation and shrinkage of mitochondria membrane, an increase in cyclin B1 level, a reduced level of Plk1 and cdc20, indicating this cell death occurred through caspase-independent, mitotic catastrophe mechanism. In contrast, an enhanced increase in cell death by Eto/AurA or Eto/AurB combination occurred through caspase-dependent mechanism. Taken together, an inhibition of aurora kinases can be combined with conventional chemotherapy to increase the response rate in AML. Novel therapeutic strategies can be designed by considering the mechanism involved in the synergistic interaction between chemotherapy and aurora kinase inhibition.