SUMMARYThe pervasive influence of secreted Wnt signaling proteins in tissue homeostasis and tumorigenesis has galvanized efforts to identify small molecules that target Wnt-mediated cellular responses. By screening a diverse synthetic chemical library, we have discovered two novel classes of small molecules that disrupt Wnt pathway responses - whereas one class inhibits the activity of Porcupine (Porcn), a membrane-bound acyltransferase that is essential to the production of Wnt proteins, the other abrogates destruction of Axin proteins, suppressors of Wnt/β-catenin pathway activity. With these small molecules we establish a chemical genetic approach for studying Wnt pathway responses and stem cell function in adult tissue. We achieve transient, reversible suppression of Wnt/β-catenin pathway response in vivo, and establish a mechanism-based approach to target cancerous cell growth. The signal transduction mechanisms shown here to be chemically tractable additionally contribute to Wnt-independent signal transduction pathways and thus could be broadly exploited for chemical genetics and therapeutic goals.

SUMMARY Recognition of the multiple roles of Hedgehog signaling in cancer has prompted intensive efforts to develop targeted pathway inhibitors. Leading inhibitors in clinical development act by binding to a common site within Smoothened, a critical pathway component. Acquired Smoothened mutations, including SMOD477G, confer resistance to these inhibitors. We report here that itraconazole and arsenic trioxide, two agents in clinical use that inhibit Hedgehog signaling by mechanisms distinct from that of current Smoothened antagonists, retain inhibitory activity in vitro in the context of all reported resistance-conferring Smoothened mutants and GLI2 overexpression. Itraconazole and arsenic trioxide, alone or in combination, inhibit the growth of medulloblastoma and basal cell carcinoma in vivo, and prolong survival of mice with intracranial drug-resistant SMOD477G medulloblastoma.

NF-κB is a master regulator of inflammation and has been implicated in the pathogenesis of immune disorders and cancer. Its regulation involves a variety of steps, including the controlled degradation of inhibitory IκB proteins. In addition, the inactivation of DNA-bound NF-κB is essential for its regulation. This step requires a factor known as copper metabolism Murr1 domain-containing 1 (COMMD1), the prototype member of a conserved gene family. While COMMD proteins have been linked to the ubiquitination pathway, little else is known about other family members. Here we demonstrate that all COMMD proteins bind to CCDC22, a factor recently implicated in X-linked intellectual disability (XLID). We showed that an XLID-associated CCDC22 mutation decreased CCDC22 protein expression and impaired its binding to COMMD proteins. Moreover, some affected individuals displayed ectodermal dysplasia, a congenital condition that can result from developmental NF-κB blockade. Indeed, patient-derived cells demonstrated impaired NF-κB activation due to decreased IκB ubiquitination and degradation. In addition, we found that COMMD8 acted in conjunction with CCDC22 to direct the degradation of IκB proteins. Taken together, our results indicate that CCDC22 participates in NF-κB activation and that its deficiency leads to decreased IκB turnover in humans, highlighting an important regulatory component of this pathway.

Suppression of oncogenic Wnt-mediated signaling holds promise as an anti-cancer therapeutic strategy. We previously reported a novel class of small molecules (IWR-1/2, Inhibitors of Wnt Response) that antagonize Wnt signaling by stabilizing the Axin destruction complex. Herein, we present the results of structure/activity relationship studies of these compounds.

The Hedgehog (Hh) and Wnt signal transduction pathways are master regulators of embryogenesis and tissue renewal and represent anticancer therapeutic targets. Using genome-wide RNA interference screening in murine cultured cells, we established previously unknown associations between these signaling pathways and genes linked to developmental malformations, diseases of premature tissue degeneration, and cancer. We identified functions in both pathways for the multitasking kinase Stk11 (also known as Lkb1), a tumor suppressor implicated in lung and cervical cancers. We found that Stk11 loss resulted in disassembly of the primary cilium, a cellular organizing center for Hh pathway components, thus dampening Hh signaling. Loss of Stk11 also induced aberrant signaling through the Wnt pathway. Chemicals that targeted the Wnt acyltransferase Porcupine or that restored primary cilia length by inhibiting the tubulin deacetylase HDAC6 (histone deacetylase 6) countered deviant pathway activities driven by Stk11 loss. Our study demonstrates that Stk11 is a critical mediator in both the Hh and the Wnt pathways, and our approach provides a platform to support the development of targeted therapeutic strategies.