Waiter Rule br Conflict of interest br Supplementary

Conflict of interest
Supplementary methods and materials
Acknowledgments This work was supported by an Early Career Researcher grant awarded by the Institute of Health and Biomedical Innovation to IMW and Queensland University of Technology, and grants awarded to SS by the Prostate Cancer Foundation of Australia and the Australian Prostate Cancer Research Centre-Queensland. JL was supported by an Australian Postgraduate Award granted to her from the Australian Government, Department of Industry, Innovation Science, Research and Tertiary Education. We would like to thank Dr Samantha Stebhens for help using the NIS-Elements program.
Introduction Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by BCR-ABL fusion protein, which generates a constitutively activated tyrosine kinase and oncogenicity. The tyrosine kinase inhibitor, imatinib (IM, STI571, Gleevec; Novartis, Basel, Switzerland) has dramatically changed the treatment of chronic phase (CP) CML [1], [2] and the 5-year survival rate has improved significantly [3], [4]. However, some patients in the CP and a substantial proportion of patients in accelerated phase (AP) and blast crisis (BC) develop resistance to the drug, with remissions usually lasting only 6 to 12months [5], [6]. Hitherto, the loss of sensitivity to IM has been attributed to the substitution of amino Waiter Rule in the kinase domain of BCR-ABL protein leading to mutations such as T315I and P-loop mutations [7], [8]. However, kinase domain mutations cannot explain the mechanism of drug resistance entirely [9]. The drug resistant BCR-ABL expressing cells seem to be at least in part independent of BCR-ABL kinase activity and might be attributed to other functional domains, leading to development of alternative pathways, such as small G protein signaling and Src signaling that are not targeted by imatinib [10], [11], [12]. Therefore, a strategy to overcome non-kinase independent IM resistance and developing novel promising agents is of paramount importance. EphB4, a member of the largest receptor protein tyrosine kinase family, plays important role in tumorigenesis and regulates diverse cell functions including cell-cell contact, cell adhesion, migration and repulsion [13]. It has been elucidated that EphB4 promotes cell adhesion mediated drug resistance via interplay with extracellular matrix and modulation of Rho family members [14]. However, it is unclear about whether EphB4 signaling was involved in IM resistance of CML. In this study, we discussed the role of EphB4 in IM resistance CML, furthermore, we explored the related downstream moleculars of EphB4. All the results suggested that EphB4-VAV1 may be a potential target emerging therapy.
Materials and methods
Results
Discussion EphB4 is a member of Eph receptor tyrosine kinase family which regulates a number of cellular events during embryonic development such as cell migration, repulsion versus adhesion and cell-to-cell communication [17]. The dichotomous EphB4 expression has been implicated in various tumors. The up-regulation of EphB4 expression is evident in carcinomas of prostate, ovary, lung, and head and neck [18]; whereas breast cancer is marked by it's down-regulation [19]. The role of EphB4 in IM resistance in CML is not well understood. How the EphB4 receptor contributes to IM resistance in CML? Several studies have demonstrated the ability of Eph receptors to activate different Rho-GTPases, such as RhoA and Rac1/cdc42 [20]. Rho family of small GTPases plays an essential role in IM resistance of CML through Bcr-Abl non-kinase pathways [21]. Based on this notion it can be hypothesized that EphB4 might contribute to IM resistance in CML plausibly through downstream Rho signaling, Rho-mediated actin cytoskeleton reorganization and cell adhesion to extracellular matrix probably via EphB4 located in cytomembrane. However, our results didn't support that result. We found that loss of EphB4 did cause a significant reduction of expression of VAV1, but not RhoA or Rac1. The results were inconsistent with the current study [22]. The VAV1 are guanine nucleotide exchange factors (GEFs) for Rho family GTPases that activate pathways leading to actin cytoskeletal rearrangements and transcriptional alterations. The encoded protein is important in hematopoiesis, playing a role in T-cell and B-cell development and activation [23].There are no reports about VAV1 to be related to IM resistance in CML. Our study showed EphB4 contributed to IM resistance might be through regulating VAV1, Rho A and Rac1 are not the downstream molecular of EphB4 receptor.