EPHA3 as a Novel Therapeutic Target in the Hematological Malignancies

Niamh Keane; Ciara Freeman; Ronan Swords; Francis J Giles


Expert Rev Hematol. 2012;5(3):325-340. 

In This Article

The Eph Receptor Tyrosine Kinases

The Eph family of receptor tyrosine kinases (RTKs) is the largest of this receptor class, with a total of 16 known Eph receptors in vertebrates and 14 Eph receptors identified in humans thus far.[1] Eph RTKs are classified as either A- or B-type receptors based on whether they preferentially bind ephrin ligands, which are membrane-bound by a GPI linker, or have a transmembrane link, respectively.[1] The first Eph family member was identified in vitro from and named after an e rythropoietin- p roducing human h epatocellular carcinoma cell line.[2] The structure of these RTKs is well conserved with similar extracellular and cytoplasmic domains.[3] The extracellular domain consists of an N-terminal globular ephrin-binding domain, a cysteine-rich linker region (recently characterized as comprising a sushi or complement control protein domain and EGF-like domain) as well as two fibronectin III repeats.[4] The intracellular domain comprises a juxta-membrane region containing two tyrosine residues, a tyrosine kinase domain, a sterile α-motif domain and a PDZ protein-binding domain (Figure 1).[5] Following ligand engagement of the receptor, phosphorylation of tyrosine moieties on a series of downstream mediators occurs, which has a myriad signal transduction effects.[3]

Figure 1.

Structure of Eph receptor tyrosine kinase. The extracellular domain contains the ephrin ligand-binding domain through which Eph receptors communicate with neighboring cells. There are two sites within the ligand-binding domain through which the ephrin binds the Eph receptor as well as a site on the adjacent sushi domain. Together the Sushi domain and EGF-like domains comprise a cysteine-rich linker region. This region is important in mediating cluster formation with other Ephs in cis. Mutations of the ligand-binding domain or the linker region can impair the ability of Anti-EPHA3 monoclonal antibody to bind the receptor. There are two fibronectin type III repeats following this. The intracellular domain consists of a number of domains including a juxtamembrane region on which two of the tyrosine residues involved in activation of the Eph kinase following ligand binding are located. The kinase region is immediately adjacent to this. Although frequently mutated in solid tumors, there have been no reports of kinase mutations in hematologic malignancies. The third tyrosine residue required for kinase activation resides within the activation loop of the kinase domain. Sterile α-motif domain and PDZ domains are located intracellularly, and these molecules may interact with other receptors and also may serve as docking sites for signaling molecules, respectively.

What sets the Ephs apart from other RTKs is their unique method of bidirectional signaling. Following ligand binding, the ephrins (Eph receptor interacting proteins) activate signal transduction not only in the Eph-bearing cell but also in the ephrin-bearing cell.[6] This unusual process underlies the mechanisms by which Ephs fulfill their various roles in cellular motility, adhesion and repulsion (Figure 1).[5,6] Receptor clustering is crucial to the process of bidirectional signaling and, in order to initiate signaling, ephrin ligands must be membrane-bound, and ephrin cluster formation possible.[7] With the binding of Eph receptor globular domain and ephrin, ligand dimers comprising the Eph and ephrin are formed, followed by interaction between dimers at tetramerization sites initiating cluster formation.[8] Cluster assembly is propagated by the interactions between neighboring Eph receptors, with increasing signaling complex assembly.[9] Ligand-binding, sushi and fibronectin III domains on the extracellular surface are capable of sustaining these interactions[4] as are the transmembrane and sterile α-motif regions intracellularly.[8,10] Kinase activation occurs following cluster formation, with phosphorylation of the three tyrosine residues required for this activation to occur.[11] These tyrosine residues may then facilitate binding of other signaling molecules, for example, Src homology 2 protein.[12]

Eph RTKs also differ from other RTKs in that they do not chiefly target nuclear transcription via their signaling cascades but rather target the cytoskeleton to alter cell adhesion/repulsion and cell motility properties by regulating activation state of Rho or Ras family GTPases.[6] Eph receptor the activation shifts the balance of Rho family members, with resultant phosphorylation of RhoA (discussed further in next section).

Eph receptors are involved in the induction of other opposing cellular responses (e.g., increased/decreased proliferation, progression/suppression of a malignant phenotype), many of which are mediated by crosstalk with other receptors and adhesion molecules (Figure 2).[13] Eph receptors actively utilize three kinase-deficient RTKs, which may contribute to the unusual flexibility of the Eph family, allowing it to initiate antagonistic responses.[14] The kinase-dead participants in Eph signaling make the prediction of the outcome of an Eph-mediated action difficult, which may reflect the balance between signaling of catalytically potent and impotent receptors. The relative concentrations of Eph receptor subtypes within the signaling complex, for example, proportions of EphA and EphB receptors, impacts the amplitude of signaling and the outcome, with the concentration of ephrin receptors having an important effect on Eph signaling as well.[5,8] Methods involved in mediating opposing actions via bidirectional signaling are discussed in greater detail below and in Figure 2.

Figure 2.

Eph receptors mediate opposing actions. (A) Following ephrin ligand binding and signaling cluster assembly, several factors determine the outcome of signaling. The default response tends to be cell repulsion. High concentrations of ephrin ligand as depicted here supports cell repulsion. In order for this to take place, there must be cleavage of the ephrin ligand by a protease and/or Eph and ephrin endocytosis as is usually seen with EphB receptors. In the case of EPHA3, ADAM10 is located close to the Eph receptors at the cell membrane. ADAM consists of a cysteine-rich region, a disintegrin and a protease domain that is activated on signaling from the cysteine-rich region in direct contact with the ligand-binding domain of the Eph receptor. ADAM10 cleaves the ephrins in trans, thus facilitating cell repulsion. Tyrosine residue phosphorylation occurs on binding of the Eph and ephrins and activates the kinase. Activation of RhoA occurs with myriad cellular effects including stress fiber contraction and dephosphorylation of FAK, which negatively regulates integrins and the net effect is cell repulsion and motility. (B) In certain settings, the outcome of Eph and ephrin interaction is cell adhesion. Low ephrin concentration favors this outcome, as does impaired tyrosine kinase function. This can occur due to the presence of kinase null Eph receptors, mutations affecting the kinase domain or activity of PTPs, which inhibit kinase activation. Instead of RhoA activity, there is a shift toward increased activity of Rac and Cdc42 members of the Rho family. This results in cellular adhesion and, in the context of malignancy, mediates invasiveness. There is no negative regulation of FAK as was seen with RhoA activation, facilitating adhesion to stroma.

Most prior tyrosine kinase-directed drugs have focused on the targeting of a dysregulated or structurally abnormal protein on which the cell is reliant for survival. In terms of targeting of Eph receptors, most progress has been made in the area of hematological malignancies with EPHA3 emerging as a putative target. Expressed aberrantly and often mutated in a wide range of malignancies,[12] EPHA3 is thought to be structurally normal in hematological cancer models and precisely what its role(s) is in terms of tumor initiation and/or propagation is not clear.


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