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.

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.

SUMMARYDirect interactions between pro- and anti-apoptotic BCL-2 family members form the basis of cell death decision-making at the outer mitochondrial membrane (OMM). Here we report that three antiapoptotic BCL-2 proteins (MCL-1, BCL-2, and BCL-XL) found untethered from the OMM function as transcriptional regulators of a prosurvival and growth program. Antiapoptotic BCL-2 proteins engage a BCL-2 homology (BH) domain sequence found in Suppressor of Fused (SUFU), a tumor suppressor and antagonist of the GLI DNA binding proteins. BCL-2 proteins directly promote SUFU turnover, inhibit SUFU-GLI interaction, and induce the expression of the GLI target genes BCL-2, MCL-1, and BCL-XL. Antiapoptotic BCL-2 protein/SUFU feedforward signaling promotes cancer cell survival and growth and can be disabled with BH3 mimetics – small molecules that target antiapoptotic BCL-2 proteins. Our findings delineate a chemical strategy for countering drug resistance in GLI-associated tumors and reveal unanticipated functions for BCL-2 proteins as transcriptional regulators.

SUMMARY Misactivation of the seven transmembrane protein Smoothened (Smo) is frequently associated with basal cell carcinoma and medulloblastoma. Cellular exposure to secreted Hedgehog (Hh) protein or oncogenic mutations in Hh pathway components induce Smo accumulation in the primary cilium, an antenna-like organelle with mostly unknown cellular functions. Despite the data supporting an indispensible role of the primary cilium in Smo activation, the mechanistic underpinnings of this dependency remain unclear. Using a novel cell membrane impermeable Smo antagonist (IHR-1) we demonstrate that Smo supplied with a synthetic agonist or activated with oncogenic mutations can signal without ciliary accumulation. Similarly, cells with compromised ciliary Smo trafficking due to loss of the phosphatidylinositol-4-phosphate 3-kinase PI3K-C2α retain transcriptional response to an exogenously supplied Smo agonist. These observations suggest that assembly of a Smo signaling complex in the primary cilium is not a prerequisite for Hh pathway activation driven by Smo agonists or oncogenic Smo molecules.

Isolated from the sponge Terpios hoshinota that causes coral black disease, nakiterpiosin was the first C-nor-D-homosteroid discovered from a marine source. We provide in this account an overview of the chemistry and biology of this natural product. We also include a short history of the synthesis of C-nor-D-homosteroids and the results of some unpublished biological studies of nakiterpiosin.

Mutations in components of the Hedgehog (HH) signal transduction pathway are found in the majority of basal cell carcinoma (BCC) and medulloblastoma incidents. Cancerous cells with intrinsic or acquired resistance to antagonists targeting the seven transmembrane effector Smoothened (SMO) frequently invoke alternative mechanisms for maintaining deviant activity of the GLI DNA binding proteins. Here we introduce a chemical agent that simultaneously achieves inhibition of SMO and GLI activity by direct targeting of the SMO heptahelical domain and the GLI-modifying enzymes belonging to the histone deacetylase (HDAC) family. We demonstrate a small molecule SMO-HDAC antagonist (IHR-SAHA) retains inhibitory activity for GLI transcription induced by SMO-dependent and -independent mechanisms frequently associated with cancer biogenesis. Synthetic combinatorial therapeutic agents such as IHR-SAHA that a priori disable cancer drivers and anticipated mechanisms of drug resistance could extend the duration of disease remission, and provide an alternative clinical development path for realizing combinatorial therapy modalities.Cellular response to the secreted HH proteins is initiated upon their binding to the multi-pass protein Patched 1 (PTCH1), a suppressor of the seven transmembrane receptor Smoothened (SMO) 1 . Activated SMO promotes SUFU disassociation from the GLI DNA binding proteins thus licensing them for gene transcriptional activation 2,3 . Deviant HH pathway activity associated with several cancers including medulloblastoma (MB) and basal cell carcinoma (BCC) is commonly induced by mutations in PTCH1 4,5 . SMO antagonists that are FDA-approved for the management of metastatic BCC (Vismodegib and Sonidegib) are able to restore homeostatic levels of signaling and blunt tumor growth 6 .Despite an impressive initial response in some metastatic BCC patients, durable tumor growth suppression by SMO antagonists has been elusive and few treatment options that are available to patients after progression. Yet, the majority of the tumors that re-emerge are likely to be still dependent upon GLI transcriptional activity as determined by the appearance of mutations in SMO that prevent drug binding 7-11 , kinase-dependent mechanisms promoting sustained GLI activity in the absence of SMO input 12,13 , or GLI2 gene amplification 8,14 . Thus, agents that disrupt GLI activity have broader indications than those targeting SMO in HH-associated cancers particularly in cases of drug resistance.A number of strategies for disrupting GLI activity have been evaluated including those that promote GLI protein turn-over such as arsenic trioxide 15,16 or GANT61 17 , 18 , or have limited mechanistic accounting 19 . The activity of GLI proteins also appear to be blunted by their acetylation thus offering opportunities for disabling GLI activity by blocking GLI deacetylases 20 . This strategy appears to be useful in blocking the growth of medulloblastomas in preclinical models of the disease 21 .We had previously described a symmetric molecule w...

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