The biological rationale for drugging Wnt signalling in cancers that possess pathway-activating mutations is certainly compelling. Nevertheless, the tractability of developing a rational drug is gated by certain criteria. First of all, we must identify a positive acting target in the pathway that is amenable to drug inhibition. Historically, small-molecule inhibitors have enjoyed success in targeting ion channels, G-protein-coupled receptors, proteases and, more recently, kinases. Although β-catenin is the most salient positive acting element in the Wnt cancer pathway, it bears no relation to these legacy targets, and direct inhibition would likely involve blocking its interaction with the TCF/LEF transcription factors. Pharmacological disruption of protein–protein interactions with small-molecule inhibitors is not without precedent, but it is rare, difficult and highly dependent upon the specifics of the structural interface one wishes to disrupt. This of course relies upon the detailed structural information, which exists in abundance for β-catenin, including complexes with several of its partners (reviewed in Xu and Kimelman, 2007).
Targeting β-catenin protein–protein interactions
From a drug perspective, the β-catenin–TCF structural interface appears daunting. Approximately 70% of the β-catenin amino-acid sequence constitutes a core superhelical region consisting of 12 armadillo repeats of 42 amino acids each. Each repeat is comprised of three helices, and together these repeats form a large positively charged groove (Huber et al, 1997). About 51 N-terminal residues of the TCF sequence occupy this groove, forming three modules of contact defined by a β-hairpin, an extended region and an α-helix (Graham et al, 2002). Although not easily recognized by sequence, cadherin, APC and ICAT, another inhibitor of β-catenin, share with TCF a chemically conserved motif within its extended region, such that the four proteins bind competitively to β-catenin (Eklof Spink et al, 2001; Huber and Weis, 2001; Graham et al, 2002; Xing et al, 2003). All four binding partners form salt bridges with lysines 312 and 435 in the armadillo repeats. Moreover, Axin and TCF overlap in their binding to β-catenin armadillo repeats 3–4, both using a single α-helix for this contact. Thus, any compound intended to disrupt the TCF–β-catenin interaction must spare the disruption of these overlapping binding partners, three of which have been classified as human tumour suppressors. Disruption of β-catenin–cadherin binding in normal intestinal epithelium has been tested in vivo by ectopic expression of an interfering fragment of cadherin. These mice developed intestinal inflammation that led to neoplasia (Hermiston and Gordon, 1995).
The extensive interaction of β-catenin with TCF, along with the overlapping binding sites with other partners, presents some serious challenges for a small-molecule inhibitor approach (Figure 2). Nevertheless, mutational analysis suggests that the appropriate compound could selectively inhibit TCF binding only. For example, the substitution H460A in β-catenin selectively hinders binding to the TCF homologue LEF1 without impairing binding to either APC or Axin (von Kries et al, 2000). β-catenin binds other important partners through contacts more discrete and perhaps more druggable than those used for TCF binding. For instance, in the nucleus β-catenin recruits various chromatin-modifying enzymes to promote gene transcription at TCF-binding sites (Willert and Jones, 2006; Mosimann et al, 2009). Among these enzymes, the p300 acetyltransferase interacts with β-catenin C-terminal sequence, including the armadillo repeat-12 (Hecht et al, 2000; Miyagishi et al, 2000; Daniels and Weis, 2002). The isolated helical domain of the β-catenin inhibitor ICAT effectively disrupts the p300/β-catenin complex, suggesting that this transcription-relevant portion of catenin could be targeted without impacting the binding of the tumour suppressors APC or Axin (Daniels and Weis, 2002).
Figure 2. Overlapping regions of interaction for β-catenin-binding proteins. The 12 armadillo repeats in β-catenin are depicted in orange and the generalized areas of interaction for various proteins that bind to them are shown. APC R3 is the third 20-amino-acid repeat unit in the APC protein and pR3 is the phosphorylated form. RA is the first 15-amino-acid repeat unit in APC.
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The binding of bcl9 to the first armadillo repeat of β-catenin also involves a far more limited interface than that defined for the TCF interaction (Sampietro et al, 2006). A single helix comprised of a couple dozen residues in bcl9 make contacts between the second and third helix of the first armadillo domain. Half of the bcl9 helix forms hydrogen bond salt bridges with β-catenin while the remaining half binds largely through hydrophobic interactions. Unfortunately, a deep pocket, typically amenable to drug binding, is not apparent in this structure. It also remains unclear whether the disruption of this interaction would be beneficial in cancer therapy. While bcl9, and its associate pygopus, appear critical for nuclear armadillo signalling in Drosophila, it plays a more limited role in mice (Jessen et al, 2008). Importantly, intestinal adenocarcinomas, driven by aberrant Wnt signalling, occur in mice devoid of intestinal bcl9, and its homologue bcl9l, at a frequency equal to that in wt mice (Deka et al, 2010). Based on gene signature analysis, the authors speculated that bcl9 null tumours possessed fewer stem cell-like characteristics and therefore could represent a less aggressive cancer than the wt tumours. Although upstream of β-catenin, the dishevelled (DVL) PDZ domain, which interacts with FZD, is potentially amenable to small-molecule disruption. This was demonstrated with Dvl PDZ-binding peptide ligands that when internalized inhibited Wnt signalling (Zhang et al, 2009). Small molecules that target this interface have also been reported (Fujii et al, 2007; Grandy et al, 2009).
Targeting kinases in the Wnt pathway
On a wish list of oncology targets, a positively acting kinase with an oncogenic mutation would rank well above any protein–protein interaction. Unfortunately, none exist in the Wnt cancer pathway. In its absence, a druggable enzyme acting downstream of β-catenin might suffice. Accordingly, Firestein et al (2008) identified the CDK8 kinase as important for both colon cancer cell viability and oncogenic Wnt signalling. Moreover, the cdk8 gene undergoes copy number gain in some colon cancers. Although this work defines CDK8 as important for β-catenin-dependent transcription, CDK8 has a broader role as a part of the Mediator complex, which is required for activator-dependent transcription by Pol II (Conaway and Conaway, 2011). Considered in this context, CDK8 is clearly not dedicated to Wnt signalling and is also required for the expression of many non-Wnt targets, including activation of the p21 gene by the p53 tumour suppressor (Donner et al, 2007). Moreoever, CDK8 has roles outside of its participation in Mediator. Nevertheless, based on its role in Wnt signalling, the amplification of its gene in colon cancer, and the ability of kinase-active, but not kinase-dead, CDK8 to transform immortalized cells, it remains an attractive target.
Casein kinase II could also be considered as a target downstream of β-catenin. Although several studies had defined a positive role for CKII in Wnt signalling, the Jones lab localized this kinase to Wnt target genes where it enhanced transactivation by enforcing the interaction of β-catenin with LEF1 (Wang and Jones, 2006). Again, inhibition of this target would likely elicit physiological effects well beyond those associated with Wnt signalling alone. The casein kinase I family has also been implicated in Wnt signalling, however, in both negative and positive directions. The phosphorylation of the LRP5/6 Wnt coreceptors by CKIγ supports receptor activation, whereas phosphorylation of β-catenin by CK1α primes it for proteolytic destruction (Liu et al, 2002; Davidson et al, 2005). Unexpectedly, the Lee lab found a small-molecule allosteric activator of CKIα in an in vitro screen designed to read out β-catenin turnover (Thorne et al, 2010). The compound, pyrvinium, potently inhibits Wnt signalling even in cells mutant for the APC tumour suppressor. It remains unclear whether systemic activation of CKI represents a tractable approach to Wnt inhibition in vivo; however, this study underscores the potential of a highly unconventional mechanism, that of kinase activation, as a novel approach in cancer therapy.
The challenge of targeting Wnt signalling with small molecules could provide some incentive for exploring unconventional approaches. Among these, lytic viruses engineered to replicate selectively in the background of enhanced β-catenin signalling have been developed. Replicating adenoviruses, in which the expression of essential viral antigens are placed under control of the TFC4 promotor, have demonstrated selective replication in cells with enhanced Wnt signalling (Brunori et al, 2001). Permutations on this approach include insertion of transgenes into the viral genome coding for proteins that render the infected cell susceptible to a prodrug or radionuclide (Lukashev et al, 2005; Peerlinck et al, 2009). Directly eliminating β-catenin by mRNA interference is also conceptually appealing. Silencing of β-catenin by transfection or local administration of small interfering RNA retards the growth of preclinical tumour models (Zeng et al, 2007; Ashihara et al, 2009; Yeung et al, 2010). However, systemic delivery of siRNA to tumours remains elusive. Interference with Wnt signalling could also be approached orthogonally by stimulating pathways that inhibit the Wnt pathway. For example, the orphan nuclear receptor RORα binds to and represses β-catenin-dependent gene activation (Lee et al, 2010).