Targeting EPHA2 with Kinase Inhibitors in Colorectal Cancer

The ephrin type‐A 2 receptor tyrosine kinase (EPHA2) is involved in the development and progression of various cancer types, including colorectal cancer (CRC). There is also evidence that EPHA2 plays a key role in the development of resistance to the endothelial growth factor receptor (EGFR) monoclonal antibody Cetuximab used clinically in CRC. Despite the promising pharmacological potential of EPHA2, only a handful of specific inhibitors are currently available. In this concept paper, general strategies for EPHA2 inhibition with molecules of low molecular weight (small molecules) are described. Furthermore, available examples of inhibiting EPHA2 in CRC using small molecules are summarized, highlighting the potential of this approach.


Introduction
For more than two decades, inhibition of kinases has been an established strategy in the treatment of various human diseases.The number of approved inhibitors supports this statement: by the end of 2022, 72 small-molecule inhibitors have been approved by the FDA for the treatment of oncological and inflammatory diseases. [1]However, these inhibitors are only focused on about two dozen target proteins, [1] excluding large areas of the kinome of successful kinase inhibitor drug development. [2]This shortcoming is striking because several of these kinase families, including the EPHrin (EPH) receptors, exhibit promising pharmacological potential.
The EPHrin family represents the largest group within the receptor tyrosine kinase (RTK) families.By interacting with the membrane-bound ephrin ligands, the EPH/ephrin system regulates various physiological and pathological processes, during development and after birth, including the immune response, angiogenesis, inflammation, atherosclerosis and cancer through cell-to-cell communication and bidirectional signaling. [3,4]sed on sequence conservation and binding affinities towards the ephrin ligands (ephrin A and B ligands differ particularly in the way they are anchored to the cell membrane, Figure 1), the receptors are divided into two subclasses (EPHA and EPHB). [5]he EPH receptors are transmembrane receptors, consisting of different subdomains (Figure 1). [6]Since both EPH receptors and ephrin ligands are cell bound, activation requires either cell-to-cell proximity (trans-activation) or can occur in the same cell expressing both molecules (cis-activation). [7]For EPH receptor inhibition, the extracellular ephrin ligand-binding domain (LBD) and the intracellular kinase domain hold particular importance.

Role of EPHA2 in Colorectal (CRC) and Other Cancer Types
Several EPHs contribute to the development of infectious diseases and cancer. [8]Here, we will focus on the role of the kinase EPHA2, which is overexpressed in glioblastoma, [9] breast [10] and colorectal cancer (CRC). [11]There is also evidence of EPHA2 involvement in other cancer types (Figure 2). [12]omprehensive reviews of the role played by EPHA2 in various cancer types have been published elsewhere. [13,14]oteworthy, high expression levels of EPHA2 are linked to poor patient prognosis, highlighting the therapeutic potential of EPHA2 inhibition. [13]EPHA2, however, is frequently coexpressed with other A and B-type EPH receptors and ligands in cancer and normal cells, each receptor/ligand complex displaying distinct or overlapping activities. [7]Also, the degree and type of receptor activation may differ depending on the size of the EPH receptor complexes and whether activation occurs from trans or cis interactions.This adds complexities to the system, as exemplified by the disparate cellular responses to EPHA2-versus EPHA4-activation by ephrinA5, leading either to cell adhesion or collapse, respectively. [15]RC is the second most common cause of cancer deaths worldwide and incidence rates of CRC correlate with the rising socioeconomic development of countries.[20] In the last few years the involvement of EPHA2 in CRC has been studied intensively.Noteworthy, certain viral infections can increase the risk of developing particular cancer types.EPHA2 is an important entry receptor for Epstein-Barr virus (EBV, which infects most adults worldwide) [21] and for Kaposi's sarcomaassociated herpesvirus (KSHV). [22]Recent studies established a link between EBV infection and the development of a lymphoepithelioma-like case of colon carcinoma [23] and with the induction of genomic instability in distinct cancer types.[24] Several studies have observed high-level expression of EPHA2 at different stages of CRC.[11,25] Especially in stage II/III CRC, EPHA2 expression is a marker of poor patient prognosis.[11] Noteworthy, genetic mutations in EPHA2 are rare (< 5 %; the COSMIC and TCGA databases).[26] Established strategies for the treatment of CRC primarily include surgical resection, chemotherapy, radiotherapy and checkpoint inhibition.[27] Targeted therapies using antibodies and small molecules are increasingly important.[27] Up to now, only a limited number of small molecule kinase inhibitors are FDA-approved for treating metastatic and advanced CRC (Figure 3): 1.The multi kinase inhibitor Regorafenib, [28] 2. A combination of the BRAF inhibitor Encorafenib with the antibody Cetuximab [29,30] and 3. A combination of the human epidermal growth factor receptor 2 (HER2) inhibitor Tucatinib and the HER2 antagonist antibody Trastuzumab (accelerated approval in January 2023).[31]

Strategies for EPHA2 Inhibition
The biological functions of EPHA2 are mediated by intracellular signaling through distinct pathways.Activation of EPHA2 induced by ephrin ligand binding, -"canonical signaling"-is mediated by autophosphorylation of tyrosine residues and kinase activity. [7]In cancer cells, constitutive EPHA2 tyrosine phosphorylation is low, and in certain settings stimulation of canonical signaling leads to attenuation of the pro-oncogenic MAPK/ERK and PI3K/Akt signaling pathways. [32]Genetic studies, however, provide evidence for a pro-tumorigenic function of tyrosine phosphorylated EPHA2 in certain cancer settings. [33]PHA2 can also signal independently of ligand engagement -"non-canonical signaling"-, which involves phosphorylation of serine and threonine residues in the linker region connecting the EPHA2 kinase and SAM domains.Multiple kinases can contribute to EPHA2 linker phosphorylation, and progressive phosphorylation of the linker region leads to EPHA2 conformational changes that impact canonical signaling.[34] Non-canonical EPHA2 signaling is often active in cancer cells, and is reported to exert pro-tumorigenic functions, particularly increased invasiveness and metastasis in cancer cells.[35,36] The complexities of EPHA2 signaling and function in cancer cells has hampered development of therapeutics in this field.
Nonetheless, different strategies have been investigated to target the EPHA2 activity (e. g. using siRNA, antibodies, peptides and small molecules). [37,38]In general, two common approaches are available for the experimental inhibition of EPHA2 by small molecules.One approach is to generate inhibitors (antibodies and small molecules) that can bind to the extracellular LBD, thereby suppressing EPHA2 interaction with the corresponding ephrin ligand (Figure 4, (1)). [37,39]18][19]  Such strategies have also been shown to block EBV infection of epithelial cells. [21,40]However, due to the large interaction surface, the design of protein-protein-inhibitors poses a challenge. [37]Molecules that have been found to interact with the LBD of EPHA2 are structurally diverse, ranging from salicylic acids [41,42] to various bile acid derivatives, including amino acid conjugates like UniPR129 [43][44][45] (Figure 5, ( 1)).The activity of the salicylic acids is controversial, as divergent results are reported for this substance class, possibly attributable to chemical instability of the molecules. [46]Of particular interest is the bile acid l-tryptophan conjugate, UniPR1331 (Figure 5, (1)), which exhibits very promising antitumoral activity in a glioblastoma xenograft model. [47]he repurposing of known compounds also succeeded.For example, the farnesoid X receptor agonist GW4064, as well as other derivatives, inhibit EPHA2 tyrosine phosphorylation (Fig- ure 5, (1)). [48,49]The approved α1-adrenergic receptor antagonist Doxazosin is an agonistic modulator of EPHA2, suppressing the downstream Akt and ERK kinase activities (Figure 5, (1)). [50]n addition to small molecule approaches, there are several peptidomimetic reagents using a similar inhibition strategy.Studies that investigated the effects of agonistic peptide-drug conjugates (PDC) or peptides containing non-natural amino acids resulted in promising compounds showing affinities in the nanomolar range towards EPHA2 (like peptide 135H11). [51,52]nother approach is to target EPHA2 signaling (Figure 4, ( 2)).However, despite the successful development of many tyrosine kinase inhibitors for the treatment of different cancers and rising interest in the role of EPHA2 in cancer development and progression, the number of inhibitors specifically designed to target the kinase domain of this receptor is limited.Along with the micromolar binders, which were developed based on screening campaigns of substance libraries (e. g. catechol, [53] and quinazoline derivatives [54] ), nanomolar inhibitors (like GLPG1790 [55] and ALW-II-41-27 [56] ) were also successfully developed. [57]Both GLPG1790 (chemical structure undisclosed) and ALW-II-41-27 showed promising effects in different cancer xenograft models (Figure 5, (2)). [58,59]everal studies examining off-target effects of tyrosine kinase (TK) inhibitors identified the applicability of known compounds, like Dasatinib, [60] for EPHA2 inhibition.S. Heinzlmeir et al. analyzed more than 230 clinically evaluated kinase inhibitors using a chemical proteomics approach (Kinobeads Assay), resulting in 24 compounds exhibiting sub-micromolar affinities for EPHA2. [61]Following this study, the effect of the EPHB4 TK inhibitor NVP-BHG712 on the kinase EPHA2 was elucidated. [62,63]Additional derivatization studies have been performed for both Dasatinib and NVP-BHG712 to generate further optimized EPHA2 inhibitors (Figure 5, (2)). [62,64,65]

Effects of EPHA2 Inhibition by Small Molecules in CRC
There are already a handful of studies showing preliminary but promising effects of EPHA2 inhibition in CRC using small molecules.The tyrosine kinase inhibitor NVP-BHG712 and its derivatives reduce cell proliferation and induce cell death in several human CRC cell lines. [63,64]The structural isomer of NVP-BHG712, NVPiso, reduces the growth of human CRC (Colo205 and HT-29 cell lines) in a xenograft mouse model. [63][64] Since many cancer types, including CRC usually express multiple A-and B-type EPH receptors, it is difficult to assign a specific EPH target when using NVP-BHG712, NVPiso and related compounds.
Similar effects have been observed with the pan-EPH inhibitor GLPG1790, which reduces EPHA2 serine phosphorylation in human CRC cells (HTC116 and HTC15 cell lines) associated with diminished cell proliferation and viability by inducing cell-cycle arrest. [66]69] Already in 2013, A. Strimpakos et al. revealed that a high expression level of the EPHA2 receptor is associated with poor patient responses to Cetuximab-based therapy. [67]y applying the EPHA2 TK inhibitor ALW-II-41-27, G. Martini et al. observed a reduction of colorectal cancer cell growth and induction of apoptosis.In addition, these authors observed that ALW-II-41-27 reverts the intrinsic (HTC15 cell line) and the acquired (SW48-CR cell line) resistance to Cetuximab in vitro and in vivo models of human CRC.Furthermore, the combination of ALW-II-41-27 and Cetuximab resulted in synergistic inhibition of proliferation, and increase in apoptosis and G 1 -G 2 cell-cycle phase arrest. [70]. Torlot et al. observed overexpression of EPHA2 (and low levels of ephrinA1) in Cetuximab-resistant CRC cell lines using a LC-MS/MS-based proteomics approach.Inhibition of EPHA2 (e. g. by Dasatinib) reduced colorectal cancer cell motility.It did not, however, restore Cetuximab sensitivity and its reductive effect on cell proliferation.Therefore, EPHA2 was characterized as a driver of migration in Cetuximab-resistant CRC cells and a potential second-line therapeutic option.[71] All these findings highlight the significant potential of EPHA2 inhibition by small molecules in the treatment of CRC.However, further studies are needed to unambiguously elucidate the role of EPHA2 in CRC and in the development of drug resistance.

Summary and Outlook
The recent findings on EPHA2 inhibition in CRC using small molecules are very promising and underline the clinical potential of targeting this kinase.Besides the direct impact of EPHA2 inhibitors on CRC cell functions, particularly proliferation as well as viability in vitro, these molecules also exhibit promising effects on reducing tumor growth in xenograft mouse models.Particularly interesting is the evidence for the contribution of EPHA2 in resistance development to the antibody Cetuximab.Initial studies indicate that the sensitivity towards Cetuximab could be restored by inhibiting EPHA2 with ALW-II-41-27.These results may have a significant and direct impact on CRC treatment in the future.
In summary, the role of EPHA2 in CRC is not yet completely elucidated, but there is evidence that EPHA2 is an important player in the development, progression, and drug resistance mechanisms in CRC.Several studies have provided initial evidence that inhibition of EPHA2 using small molecule is a promising approach for the treatment of CRC.The development of additional EPHA2 inhibitors will support future fundamental research campaigns to clarify the role of EPHA2 in CRC and may additionally have a potential clinical use in treating CRC patients.

Figure 1 .
Figure 1.Schematic representation of the EPH/ephrin system.The ephrin ligands are different in the way they interact with the cell membrane.The EPH receptor consists of an extracellular, transmembrane, and intracellular region.The intracellular region includes a flexible juxtamembrane domain, a kinase domain, a SAM domain and a PDZ domain.The extracellular region consists of two fibronectin repeat units and a ligand binding domain for interaction with ephrin ligands.The EPH/ephrin system is characterized by bidirectional signaling (forward and reverse signaling, ligands are also capable of inducing a response in their cell).(GPI: glycosylphosphatidylinositol, RBD: receptor binding domain, SAM: sterile alpha motif)

Figure 4 .
Figure 4. Interaction of small molecules with the extracellular ligand binding domain (1) and the intracellular kinase domain (2) of EPHA2.Chemical structures of the corresponding inhibitors are depicted in Figure 5.

Figure 5 .
Figure 5.Chemical structures of inhibitors binding to the extracellular ligand binding domain (1); Chemical structures of inhibitors binding to the intracellular kinase domain (2).