YAP1 is essential for malignant mesothelioma tumor maintenance.

Malignant pleural mesothelioma, a tumor arising from the membrane covering the lungs and the inner side of the ribs, is a cancer in which genetic alterations of genes encoding proteins that act on or are part of the Hippo-YAP1 signaling pathway are frequent. Dysfunctional Hippo signaling may result in aberrant activation of the transcriptional coactivator protein YAP1, which binds to and activates transcription factors of the TEAD family. Recent studies have associated elevated YAP1 protein activity with a poor prognosis of malignant mesothelioma and its resistance to current therapies, but its role in tumor maintenance is unclear. In this study, we investigate the dependence of malignant mesothelioma on YAP1 signaling to maintain fully established tumors in vivo. We show that downregulation of YAP1 in a dysfunctional Hippo genetic background results in the inhibition of YAP1/TEAD-dependent gene expression, the induction of apoptosis, and the inhibition of tumor cell growth in vitro. The conditional downregulation of YAP1 in established tumor xenografts leads to the inhibition of YAP1-dependent gene transcription and eventually tumor regression. This effect is only seen in the YAP1-activated MSTO-211H mesothelioma xenograft model, but not in the Hippo-independent HCT116 colon cancer xenograft model. Our data demonstrate that, in the context of a Hippo pathway mutated background, YAP1 activity alone is enough to maintain the growth of established tumors in vivo, thus validating the concept of inhibiting the activated YAP1-TEAD complex for the treatment of malignant pleural mesothelioma patients.

High-affinity Src-SH2 ligands which do not activate Tyr(527)-phosphorylated Src in an experimental in vivo system.

The Src-SH2 domain has been determined to play a key role in many signaling pathways, especially in osteoclast-mediated bone resorption. Therefore, non-peptidic small molecules, mimicking the natural pYEEI peptide ligand, have been designed, to inhibit SH2-mediated protein-protein interactions and provide therapeutic treatment of certain diseases such as osteoporosis. However it has been shown in vitro that phosphopeptidic ligands of the SH2 domain are able to increase Src kinase activity by disrupting the intramolecular interactions between the Tyr(521)-phosphorylated C-terminal tail and the SH2 domain, thereby inducing a change from a "closed" inactive to an "open" active conformation of Src. Thus it was not clear whether non-peptidic ligands would limit their action to the inhibition of the signaling cascade by interfering with the intermolecular SH2 binding, or would activate the enzyme as do phosphopeptides. To address this question we have investigated the effects of a series of both peptidic and non-peptidic ligands of the SH2 domain on Src kinase activation, both in vitro in an ELISA based assay and in vivo using csk and src double transformed Schizosaccharomyces pombe. We found that, in the peptide series, the extent of c-Src activation is directly correlated to the respective binding affinity for Src-SH2. By contrast such correlation is not valid for non-peptidic ligands, some high-affinity SH2 binders showing no detectable Src activation in vivo. These results have significant implications for the design of SH2 binders, as they allow a way to inhibit Src-SH2-mediated signal transduction in target cells, without activating Src in non-target cells, thereby reducing the possibility of side effects.

SAR and X-ray. A new approach combining fragment-based screening and rational drug design: application to the discovery of nanomolar inhibitors of Src SH2.

(pp60)Src is a protein involved in signal transduction and is mainly expressed in neurones, platelets, and osteoclasts. Its precise biological role was recently discovered with the KO experiments by Soriano that gave rise to no other apparent phenotype than osteopetrosis, a disease resulting in excedent bone formation. The SH2 domain of the Src family specifically recognizes a sequence of tetrapeptide featuring a phosphotyrosine and a lipophilic aminoacid at the +1 and +3 positions. Recently we engaged in the search for SH2 ligands via modular peptidomimicry of this tetrapetide. This gave rise to several families of nanomolar inhibitors; the best one incorporated a caprolactam scaffold, a biphenyl moiety, and a phosphotyrosine. However, these inhibitors still incorporated the phosphate group that confers good binding affinity to the protein. Phosphates have undesirable features for drug candidates, namely, high rate of hydrolysis of the phosphate group by phosphatases and high charge content precluding cell penetration. Therefore, while searching for optimal non-peptide ligands for Src SH2, we looked for phosphate replacements. For this, we have designed an SAR by fragment crystallography approach. The start of this work resulted from two experimental observations. First, the fact that phenyl phosphate itself displayed detectable binding affinity for Src SH2 permitted us to perform a screening of small aromatic compounds as phenyl phosphate surrogates. Second, the obtention of large Src SH2 crystals displaying a channel large enough for soaking purposes allowed structure determination of over 40 of these small aromatic compounds bound in the phosphotyrosine binding pocket. This search and the way it gave rise to low nanomolar range Src SH2 inhibitors devoid of phosphate groups will be the subject of the present paper.