Aurora Kinase a: A New Component of Imatinib Resistance in Chronic Myeloid Leukemia

Imatinib (IM) targets the constitutively active BCR-ABL1 tyrosine kinase (TK) in chronic myeloid leukaemia (CML) and has become standard treatment based on good responses achieved in clinical trials. However, IM resistance can occur through several mechanisms including BCR-ABL1 kinase domain mutations, amplification, overexpression and clonal evolution.

The discovery that Aurora kinases (AK) were abnormally expressed in malignancies including leukaemia prompted the development of agents that inhibit their activity. Aurora kinase A (AKA) is a central mitotic regulator necessary for mitotic entry, mitotic spindle assembly and accurate chromosome separation. The therapeutic potential of specifically targeting AKA activity as an anticancer strategy has not been rigorously investigated because all of the agents previously designed to target AKs have significant off-target effects on other family members and/or BCR-ABL1 kinase activity. However, the role of AKA in chemoresistance of CML is not well understood.

In this study we investigated the mechanism of action of AKA in CML in vitro and ex vivo models (i.e. K562 cell lines and CD34+ cells derived from bone marrow aspirates of both CML at the clinical diagnosis and in mononuclear cell fraction of IM-resistant patients). Moreover, the role of two major players (i.e. FOXM1 and Polo-like kinase 1 (PLK1) a ser-thr kinase involved in G2/M progression) in the AKA signalling pathway has been investigated.

First, we investigated AKA expression and activity by Western Blot and immunoprecipitation analyses. CD34+ cells from peripheral blood aphereses of hematologically healthy donors, pooled to avoid individual differences, were used as normal controls. Informed consent was obtained by all participants before their admittance to the study. Our data showed an over-expression and hyper-phosphorylation of AKA, FOXM1 and PLK1 in CD34+ cells of CML patients at clinical diagnosis compared to mononuclear cell fractions.

Our results also showed a FOXM1 increment associated with IM resistance. An IM-resistant K562 cell line (LD50 0.25 mM vs 0.026 mM of parental cells) generated in our lab exhibited FOXM1 over-expression and hyper-phosphorylation contingent upon the upstream activation of AKA and PLK1. AKA, FOXM1 and PLK1 involvement in IM resistance was observed also in clonal progenitors from bone marrow samples of CML patients who developed IM resistance independent from BCR-ABL1 point mutations. Interestingly, the putative BCR-ABL1+/CD34+ LSC compartment, which is neither dependent on BCR-ABL1 TK for proliferation and survival nor killed by IM or second generation inhibitors, showed a hyper-phosphorylation of AKA and a consequent overexpression and hyper-activation of FOXM1 and PLK1.

Taken together, these evidences may pave the way to the set up a new strategy to overcome drug resistance in CML. Moreover, our data identify a new signaling pathway involved both in drug resistance and in CD34+ cells survival, suggesting that AKA, FOXM1 and PLK1 are three very interesting druggable targets to eradicate the TKIs resistant CD34+ progenitors.

Acknowledgements: Manuela Mancini was supported by a Fondazione Umberto Veronesi Fellowship. University of Bologna (RFO funds), Ministero della Pubblica Istruzione e della Ricerca (PRIN funds), Umberto Veronesi Foundation, AIRC, FP7 NCS-PLT project, Progetto Regione-Università 2010-12 (L. Bolondi) and BolognaAILare acknowledged for financial support.