Ligand-dependent activation of the hedgehog (Hh) signalling pathway has been associated with tumorigenesis in a number of human tissues. Here we show that, although previous reports have described a cell-autonomous role for Hh signalling in these tumours, Hh ligands fail to activate signalling in tumour epithelial cells. In contrast, our data support ligand-dependent activation of the Hh pathway in the stromal microenvironment. Specific inhibition of Hh signalling using small molecule inhibitors, a neutralizing anti-Hh antibody or genetic deletion of smoothened (Smo) in the mouse stroma results in growth inhibition in xenograft tumour models. Taken together, these studies demonstrate a paracrine requirement for Hh ligand signalling in the tumorigenesis of Hh-expressing cancers and have important implications for the development of Hh pathway antagonists in cancer.

The Hedgehog (Hh) signaling pathway is inappropriately activated in certain human cancers, including medulloblastoma, an aggressive brain tumor. GDC-0449, a drug that inhibits Hh signaling by targeting the serpentine receptor Smoothened (SMO), has produced promising anti-tumor responses in early clinical studies of cancers driven by mutations in this pathway. To evaluate the mechanism of resistance in a medulloblastoma patient who had relapsed after an initial response to GDC-0449, we determined the mutational status of Hh signaling genes in the tumor after disease progression. We identified an amino acid substitution at a conserved aspartic acid residue of SMO that had no effect on Hh signaling but disrupted the ability of GDC-0449 to bind SMO and suppress this pathway. A mutation altering the same amino acid also arose in a GDC-0449–resistant mouse model of medulloblastoma. These findings show that acquired mutations in a serpentine receptor with features of a G protein–coupled receptor can serve as a mechanism of drug resistance in human cancer.

The activation of the AKT/protein kinase B kinases by mutation of the PTEN lipid phosphatase results in enhanced survival of a diversity of tumors. This resistance to apoptosis is partly accomplished by the inhibition of genetic programs induced by a subfamily of forkhead transcription factors including AFX. Here we describe an AFX-regulated pathway that appears to account for at least part of this apoptotic regulatory system. Cells induced to synthesize an active form of AFX die by activating the apoptotic death pathway. An analysis of genes regulated by AFX demonstrated that BCL-6, a transcriptional repressor, is up-regulated ϳ4 -7-fold. An examination of the BCL-6 promoter demonstrated that AFX bound to specific target sites that could activate transcription. BCL-X L , an anti-apoptotic protein, contains potential BCL-6 target sites in its promoter. An analysis of endogenous BCL-X L levels in AFX-expressing cells revealed enhanced down-regulation of the transcript (ϳ1.3-1.7-fold) and protein, and BCL-6 directly binds to and suppresses the BCL-X L promoter. Finally, macrophages isolated from BCL-6؊/؊ mice show enhanced survival in vitro. These results suggest that AFX regulates apoptosis in part by suppressing the levels of anti-apoptotic BCL-XL through the transcriptional repressor BCL-6.The resistance of tumors to diverse apoptotic stimuli is of major clinical importance. A large number of recent studies has highlighted the significance of hyperactivation of the PI3K 1 pathway to the insensitivity of late stage tumors to chemotherapy and radiation treatments (1-6). The induction of this pathway by growth factor or oncogenic stimuli results in the production of three phosphorylated phosphatidylinositol lipids, which act as binding sites for the pleckstrin homology domains of the downstream kinases, PDK-1 and AKT/PKB (7-13). The membrane colocalization of these kinases results in the phosphorylation of a specific activation site in AKT/PKB by PDK-1 (7,8,(13)(14)(15). Importantly, this pathway is reversed by the lipid phosphatase activity of PTEN, a tumor suppressor that is homozygously deleted in a variety of late stage tumors (2-6). Whereas a number of phosphorylation targets has been described for the AKT/PKB kinases, both biochemical and genetic evidence support a subset of forkhead transcription factors including FKHR, FKHR-L1, and AFX as being important substrates for phosphorylation mediated by these kinases (16 -20). Phosphorylation of these forkhead transcription factors by AKT/PKB kinases results in their sequestration to the cytoplasm in which they are unable to activate transcription of their nuclear targets (17)(18)(19)(20). This outcome is an important component of cell survival mediated by the PI3K pathway, because nuclear localization of these forkhead family members results in the induction of transcriptional programs that lead to rapid cell death by apoptosis (17)(18)(19)(20).Whereas it is clear that the inhibition of the apoptotic programs activated by these forkhead transcription factors is ...

Purpose: The Notch pathway plays an important role in both stem cell biology and cancer. Dysregulation of Notch signaling has been reported in several human tumor types. In this report, we describe the development of an antibody, OMP-59R5 (tarextumab), which blocks both Notch2 and Notch3 signaling. Experimental Design: We utilized patient-derived xenograft tumors to evaluate antitumor effect of OMP-59R5. Immunohistochemistry, RNA microarray, real-time PCR, and in vivo serial transplantation assays were employed to investigate the mechanisms of action and pharmacodynamic readouts. Results: We found that anti-Notch2/3, either as a single agent or in combination with chemotherapeutic agents was efficacious in a broad spectrum of epithelial tumors, including breast, lung, ovarian, and pancreatic cancers. Notably, the sensitivity of anti-Notch2/3 in combination with gemcitabine in pancreatic tumors was associated with higher levels of Notch3 gene expression. The antitumor effect of anti-Notch2/3 in combination with gemcitabine plus nab-paclitaxel was greater than the combination effect with gemcitabine alone. OMP-59R5 inhibits both human and mouse Notch2 and Notch3 function and its antitumor activity was characterized by a dual mechanism of action in both tumor and stromal/vascular cells in xenograft experiments. In tumor cells, anti-Notch2/3 inhibited expression of Notch target genes and reduced tumor-initiating cell frequency. In the tumor stroma, OMP-59R5 consistently inhibited the expression of Notch3, HeyL, and Rgs5, characteristic of affecting pericyte function in tumor vasculature. Conclusions: These findings indicate that blockade of Notch2/3 signaling with this cross-reactive antagonist antibody may be an effective strategy for treatment of a variety of tumor types. Clin Cancer Res; 21(9); 2084–95. ©2015 AACR.

Mutations in the neurofibromatosis type 2 (NF2) gene that limit or abrogate expression of functional Merlin are common in malignant mesothelioma. Merlin activates the Hippo pathway to suppress nuclear translocation of YAP and TAZ, the major effectors of the pathway that associate with the TEAD transcription factors in the nucleus and promote expression of genes involved in cell proliferation and survival. In this article, we describe the discovery of compounds that selectively inhibit YAP/TAZ-TEAD promoted gene transcription, block TEAD auto-palmitoylation, and disrupt interaction between YAP/TAZ and TEAD. Optimization led to potent analogs with excellent oral bioavailability and pharmacokinetics that selectively inhibit NF2-deficient mesothelioma cell proliferation in vitro and growth of subcutaneous tumor xenografts in vivo. These highly potent and selective TEAD inhibitors provide a way to target the Hippo-YAP pathway, which thus far has been undruggable and is dysregulated frequently in malignant mesothelioma and in other YAP-driven cancers and diseases.