At the ASCO and the AACR/NCI/EORTC meetings there was an avalanche of publicity about immunotherapies, combination therapies, mechanisms of tumor resistance and tumor genetics: all areas of intense importance and astounding progress. New therapeutic modalities, such as epigenetic regulation, also received great attention. Less noticed, but important I think, was the slow but steady progress being made toward the effective targeting of the Wnt pathway.

The Wnt pathway leading to oncogenic activation of β-catenin has been studied for decades but only now are we seeing effective means of antagonizing Wnt signaling. The importance of the Wnt pathway in oncogenesis was revealed in the course of investigation of tumor causing murine retroviruses during the 1980s and 1990s. Work in the Varmus lab and others led to the discovery that the Mouse Mammary Tumor Virus (MMTV) was oncogenic due to a proviral insertion that activated a gene called int1. Int1 was subsequently renamed Wnt1, based on homology of the protein to the Drosophila family of Wingless proteins (encoded by the Wg genes). For a beautiful review of the field see Nusse & VarmusEMBO J. 31: 2670-2684.

The Wnt proteins are secreted from cells as 350 – 400 amino acid lipid-modified glycoproteins. The lipid modifications are required for effective cell secretion and also for receptor binding. For example, the Porcupine protein, an O-acyltransferase, is required for palmitoylationthat allows efficient secretion of Wnt. All Wnt proteins (there are more than a dozen in mammals) bind to the Frizzled receptors, a large family of G-protein coupled signaling receptors (GPCRs). Frizzled is most often found in a cell surface complex with co-receptors, notably the low-density lipoprotein-related protein, LPR5/6. Binding to the receptor complex triggers complex and fascinating signaling cascades. Signaling is mediated by phosphorylation of the cytoplasmic protein Dishevelled (Dsh), that signals through several distinct activation domains. Critical to cell activation is Frizzled/LRP5/6-mediated displacement of the negative regulatory complex that includes the proteins Axin, APC, GSK3β and several others. This negative regulatory complex is called the “destruction complex” since its normal function is to degrade, via the ubquitination/proteosome pathway, the critical Wnt pathway signaling protein called β-catenin.

Accumulation of b-catenin leads to translocation from the cytoplasm into the nucleus, and interaction with the transcription factors TCF/Lef1 and the Creb-binding protein (CBP). Many genes are known to have TCF/Lef1 and CBP binding sites in their promoters and are therefore potential targets for Wnt signaling. Many of these genes in turn have been implicated in tumor genesis, growth and survival. Notable genes targeted by β-catenin signaling include c-myc, Cyclin D, c-jun, various growth factors, and many others. Both TCF-1 and Lef-1 are up-regulated in an autocrine manner, further propagating β-catenin-dependent signaling.

Evidence for the critical role of the Wnt pathway in tumor pathogenesis has come from genetic studies of the pathway’s different components. Accumulation of β-catenin has been observed in diverse cancers. Mutations associated with cancer include the loss-of-function mutations of APC in colorectal cancer that decrease the rate of β-catenin degradation.  Mutations have also been described in the β-catenin gene CTNNB1 and in the Axin gene (AXIN1), among others. The effect of the most common of these gene mutations is to prevent degradation of β-catenin. Other mechanisms of Wnt pathway regulation are described below in the context of drug development.

Critical advances in the understanding and targeting of the Wnt pathway in cancer were presented this year at the ASCO annual meeting in May and at the AACR-NCI-EORTC “Molecular Targets and Cancer Therapeutics” conference held last week in Boston. These advances specifically address the role of aberrant Wnt pathway signaling in the context of tumor cell proliferation and survival, and also in the emerging field of cancer stem cell biology. The potential of this pathway in cancer therapeutics is indicated by the appearance of pathway antagonists in biotech and pharma portfolios.  Examples are given below, and there are certainly additional efforts underway.

Several compounds have reached clinical trials, including both small molecules and biologic drugs. A leading therapeutic class is the Porcupine inhibitors, as exemplified by LGK974 from Novartis. Porcupine inhibitors reduce O-acyltransferase activity by Porcupine and thereby antagonize Wnt protein secretion. LGK974 has shown activity in preclinical tumor models and is currently in Phase 1/2 clinical trials in melanoma and breast cancer to establish dose and tolerability. Other porcupine inhibitors are in preclinical development and some of these are listed on the Wnt homepage (http://www.stanford.edu/group/nusselab/cgi-bin/wnt/smallmolecules). Preclinical data using a second porcupine inhibitor, C59, was reported at the AACR-NCI-EORTC meeting. Using this inhibitor, Wnt-dependent tumor growth was blocked in xenograft tumor models, without evidence of overt toxicity.

There are even more compounds in preclinical development that act by stabilizing the Axin protein, thereby maintaining the “destruction complex” and preventing β-catenin accumulation. One is XAV939 from Novartis, an antagonist of tankyrase (TRF1-interacting ankyrin-related ADP-ribose polymerase; TNK). TNK antagonists act by inhibiting the enzymatic activity of TNK1 and TNK2 that act to mediate Axin ubquitination and proteosomal degradation. Axin targeting is being pursued aggressively for two reasons: first Axin mutations are associated with increased levels of β-catenin in diverse cancers, including colorectal carcinomas, hepatocellular carcinomas, and medulloblastomas. Second, interesting work has suggested that Axin is the rate-limiting component of the “destruction complex” at least in some experimental systems. The Novartis compound XAV939 is one of numerous TNK1/2 inhibitors in preclinical development. Genentech has reported that its inhibitor (G007-LK) was active in models of colorectal cancer cells carrying APC loss-of-function mutations. This is a critical therapeutic profile if such inhibitors are to find wide utility. The Genentech program has been licensed to Odin for use in colorectal cancer. A recent paper presented structural models of the binding of G007-LK and a novel inhibitor WIK14 to tankyrase (Haikarainen et al. PLoS ONE 8: e65404). Programs from Kyowa Hakko Kirin and others are now visible in various publications, abstracts and patents.

Another small molecule, PRI-724, blocks the interaction of β-catenin with the transcription factor CBP to prevent pro-growth and pro-survival gene expression. PRI-724, developed by PRISM in collaboration with Eisai Pharmaceuticals, is in Phase 1 clinical trials in AML and advanced solid tumors. Inhibitors of T-NIK activity are also being advanced. T-NIK is an activating kinase for some TCF transcription factors, and appears to be required for colorectal cancer cell proliferation. Astex Pharmaceuticals (now owned by Otsuka) has an active preclinical program. Carna Biosciences presented characterization data on a tool compound at the AACR-NCI-EORTC meeting.

Anti-Frizzled receptor antibodies constitute a distinct class of Wnt pathway inhibitors. The most advanced of these, vantictumab (OMP-18R5) from OncoMed/Bayer. This antibody binds to five of the frizzled receptors, and is in Phase 1 clinical trials, with interim data reported at meetings this year. Patients with advanced, refractory solid tumors were treated with single-agent vantictumab at doses up to 15 mg/kg every three weeks. The investigators have stated that the 15 mg/kg dose maintained an efficacious exposure, based on rodent tumor models. Evidence of single-agent activity of vantictumab was noted in several neuroendocrine tumor patients. An interesting question is whether this effect on the tumors was on mechanism (i.e. due to inhibition of Wnt/Frizzled interaction) or due to effector function of the antibody, sufficient to induce cell killing at the site of antibody binding. At the AACR-NCI-EORTC meeting PD data was presented that was clearly on mechanism, based on the regulation of stem cell and differentiation genes expressed in tumor and hair follicles (sampled) and bone (inferred from blood samples). PD effects were noted at all doses examined. OncoMed and Bayer are also developing a Frizzled-Fc fusion protein, mimicking a normal means of regulation by secreted extracellular domain fragment of Frizzled proteins by cells.

The discussion of PD markers in response to Wnt pathway inhibition brings up several interesting issues. Effective inhibition of Wnt pathway signaling will potentially block normal stem cell renewal, most critically of the intestinal epithelial compartment. This single cell epidermal layer creates the mucosal barrier that maintains the sterility of the mucosal tissues lying adjacent to the lumen running from the mouth to the anus. Compromising the integrity of the mucosal epithelium can lead to toxicity ranging from gastrointestinal discomfort to more severe manifestations resulting in sepsis.  The turnover of gut epithelial cells is 4 days in human, meaning that any ablation of stem cell cycling would have an impact fairly quickly. Therefore maintaining a therapeutic window will be critical in the setting of Wnt antagonism.

An area of intense recent interest has been in the field of cancer stem cells, putatively acting not only as oncogenic progenitors but importantly as the source of resistant populations following conventional (chemo, radiation) or targeted (rational, immunotherapy) treatments. The alarming spread of highly aggressive treatment resistant cancer that occurs in many or most settings of solid tumor therapy speaks to the importance of the stem cell like properties of some cancer cells. The extent that stem cells from different tumor types are dependent on Wnt signaling has not yet been determined. The best data come from studies of colorectal cancer. Demonstration that gut stem cells and their oncogenic progeny are dependent on Wnt signaling was beautifully presented by Hans Clevers at the AACR-NCI-EORTC meeting last week (see his review at Cell 54: 274-284). Recent published data also suggest a critical role for the Wnt pathway in maintaining the complex signaling matrix required to support glioblastoma proliferation (PLoS Comput Biol. 9: e1002887).

Another interesting question with respect to Wnt pathway antagonism is where in the pathway to intervene. Upstream antagonists such as the anti-frizzled antibody or the porcupine inhibitors may not be effective in cases where the downstream components have been mutated. Therefore, such therapeutics may best be used in the context of a high β-catenin signature absent Axin or APC mutation. Downstream antagonists such as β-catenin-CBP antagonists may have potentially useful specificity but may not provide sufficient inhibition of the signaling cascade. In this context, tankyrase inhibitors appear to be sitting in the right spot. Finally, alternative pathways to β-catenin activation have been described although it is not understood yet to what extent these pathways are active in tumor biology in situ. These alternative pathways may not be targeted by current therapeutic approaches.

Regardless, the Wnt antagonism field has now grown to encompass diverse intervention points, and the first antagonists have entered clinical trials. Early signs of success, if they come, will no doubt continue to drive interest in this critical oncogenic pathway.