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  • br Materials and methods br Results br Discussion DAS


    Materials and methods
    Discussion DAS and nilotinib, as the second potent, oral generation TKIs, bind to both the active and inactive conformation of the ABL1 kinase domain, have been found to be effective in treating patients with IM resistant or intolerant CML-CP, and inhibit all tested IM-resistant mutations, except T315I [14]. Compared with nilotinib, DAS not only inhibits BCR-ABL and SRC-family kinases, but also c-KIT, PDGFR-alpha and beta, and Ephrin receptor kinases [15]. It has been suggested [10] that the overexpression of EphB4 is a new marker of IM resistance. In the findings, the determined five subjects (A–E) with resistance to DAS were not detected as having these harboring highly-resistant mutations (T315I/A, F317L/V/C and V299L). The expression of EphB4 significantly increased in A2–E2 human STF 31 with high DAS resistance but decreased in A1–E1 human cells with sensitivity to DAS. The introduction of tyrosine kinase inhibitors (TKIs)-IM has fundamentally changed the management of CML. However, once the disease has progressed to an accelerated or blast phase, there is no consensus regarding optimal therapy [16]. Presently, DAS is approved for the treatment of patients with BCR-ABL-positive CML who are resistant or intolerant to IM in the chronic, accelerated, and blast phases. The previous report showed [10] that the overexpression of EphB4 is associated with IM resistance through the RhoA mechanism in CML. It is an important pathway inducing apoptosis and mitochondrial injury for activating RhoA/ROCK1/PTEN signaling in human leukemia cells [17]. Our study found that the expressions of p-Rac1/Cdc42, ROCK1, phospho-PTEN, and MCL-1 were increased in K562-R cells and xenografts but decreased in K562-R-EphB4-sh cells and xenografts. The EphB4/RhoA overexpression induced resistance to DAS by activating the ROCK1/PTEN/MCL-1 mechanism. EphB4/Ephrins play important roles in oncogenesis and in tumor growth progression [18], [19]. Previous research demonstrated that EphB4/Ephrins promoted cell adhesion and mediated drug resistance through contact with the extracellular matrix and regulating Rho family members in the microenvironment [20]. In the study, the EphrinB2-Fc made K562-R-EphB4-sh cells restore sensitivity to DAS by stimulating the expression of EphB4. Accordingly, in the stimulation of EphrinB2-Fc, the expressions of EphB4/RhoA/MCL-1 were significantly increased in K562-R-EphB4-sh cells. In summary, the present study found that a new DAS resistance pathway of EphB4 overexpression was triggered by EphrinB2-Fc and induced resistance to DAS by activating RhoA/ROCK1/PTEN/MCL-1 signaling.
    Conflict of interest
    Funding This study was funded by the National Natural Science Foundation of China (grant number 81460027).
    Ethical approval
    Informed consent
    Introduction Eph receptors (EphA1-8,10 and EphB1-4,6) constitute the largest family of receptor tyrosine kinases, and their membrane-bound ephrin ligands are either glycophosphatidylinositol-linked A-type (ephrinA1-10) or transmembrane B-type (ephrinB1-3) proteins. Eph-ephrin interactions thus occur between apposed cells, with bidirectional signaling in some cases (1, 2). These juxtacrine cues play an integral role in normal developmental processes such as tissue patterning (3) and axonal pathfinding (4), as well as in abnormal pathological conditions such as developmental disorders and cancer (5, 6). Eph receptors are a unique class of receptor tyrosine kinases for which activity requires not only Eph dimerization and transphosphorylation but also multivalent oligomerization into higher-order clusters to initiate downstream signaling (7, 8). Furthermore, Eph receptors can exhibit homotypic and heterotypic cis-interactions in addition to intercellular trans-interactions with various Ephs and ephrins to form complex signaling clusters (2, 9). Eph signaling cluster size, composition, spatial organization, and mechanical forces have all been identified as possible modulators of Eph signaling and the resultant functional cellular outcomes (10, 11). In particular, mechanical sensitivity and spatial organization of cell surface receptors are increasingly recognized as relevant cellular stimuli (12, 13, 14, 15, 16). For instance, when EphA2-ephrin-A1 signaling cluster movement is physically restricted, proximal membrane signaling events including recruitment of the downstream signaling effector ADAM10 (17) and ephrin-A1 trans-endocytosis (18) are markedly altered.