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The Hippo pathway is an evolutionary conserved signaling network that regulates essential processes such as organ size, cell proliferation, migration, stemness and apoptosis. Alterations in this pathway are commonly found in solid tumors and can lead to hyperproliferation, resistance to chemotherapy, compensation for mKRAS and tumor immune evasion. As the terminal effectors of the Hippo pathway, the transcriptional coactivators YAP1/TAZ and the transcription factors TEAD1–4 present exciting opportunities to pharmacologically modulate the Hippo biology in cancer settings, inflammation and regenerative medicine. This review will provide an overview of the progress and current strategies to directly and indirectly target the YAP1/TAZ protein–protein interaction (PPI) with TEAD1–4 across multiple modalities, with focus on recent small molecules able to selectively bind to TEAD, block its autopalmitoylation and inhibit YAP1/TAZ–TEAD-dependent transcription in cancer.

Over the past years, the HIPPO tumor suppressor pathway and its downstream effector Yes1-associated transcriptional regulator (YAP1) present increasing interest in cancer research. The YAP1-TEAD complex regulates transcription for cell proliferation, survival, plasticity, and migration. Aberrant activation of YAP1 has been reported for multiple different cancer types. To estimate the proportion of cases where YAP1/TEAD-dependent transcription may be responsible for tumor development or evolution and evaluate the pharmacodynamics of novel TEAD-inhibitors, we have identified a transcriptional signature of YAP1 activity. The signature comprises 500 genes, positive and negative downstream effectors at about equal proportions, selected by differential expression analyses from 10 published datasets reporting, in total, 19 YAP1-activation and 18 YAP1-inhibition experiments. YAP1/TEAD-dependent transcription was scored in single samples as the difference Rp - Rn, where Rp is the mean fractional rank of the positive YAP1-effectors, and Rn, that of the negative effectors. We so scored, and called, YAP1 activation in normal tissues (GTEx collection; N~9000), in primary, recurrent, or metastatic tumors (TCGA; N~10000), and in cell lines (CCLE; N~1000) for the selection of models. We further applied the signature to follow the degree of YAP1 inhibition in cell and tumor models either after genetic knockdown of HIPPO-YAP1 pathway components or by pharmacologic inhibition using small-molecule TEAD-inhibitors. Our data confirm the robustness of the YAP1-transcription signature as a biomarker for both, tumor indication selection and pharmacodynamics of YAP1-TEAD activity. Citation Format: Emmanuel Spanakis, Loreley Calvet, Odette Dos Santos, Colette Dib, Sukhvinder Sidhu, Jurgen Moll, Laurent Debussche, Jack Pollard, Iris Valtingojer. A transcriptomic signature for measuring YAP1 activity in patient samples and tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2161.

Yes associated protein YAP1 (YAP1) is a cofactor of gene transcription and exerts its action through association with transcription factors from the TEAD family. Activity of YAP1-TEAD is tightly regulated by the HIPPO pathway and upon activation the expression of pro-survival and anti-apoptotic genes is induced. The HIPPO pathway consists of a series of Ser/Thr kinases, which lead to the phosphorylation of YAP1, and hence to its inactivation by either sequestration in the cytoplasm, or degradation. HIPPO pathway deletions and YAP1 activation are particularly prevalent (> 60 %) in patients with malignant pleural mesothelioma. We use a malignant mesothelioma tumor cell line with a HIPPO deleted genetic background (MSTO-211H, LATS1 loss) to demonstrate, that downregulation of YAP1 leads to the inhibition of YAP1-dependent gene expression, the induction of apoptosis and the inhibition of cell growth in vitro. When these cell lines are grown as an in vivo subcutaneous xenograft in mice, downregulation of YAP1 leads to the inhibition of YAP1-dependent gene expression and eventually results in the regression of established tumors. This effect is specific to YAP1 activated mesothelioma models and is not observed in the HIPPO pathway independent HCT116 colon cancer model. We show here, that YAP1 activity alone is required for tumor maintenance in vivo in the context of HIPPO pathway genetic alterations, and thus confirm the relevance of this target for a drug discovery approach. Citation Format: Loreley Calvet, Odette Dos Santos, Véronique Jean-Baptiste, Emmanuel Spanakis, Yvette Ruffin, Isabelle Sanchez, Jessica Mestadier, Stéphane Soubigou, Sylvie Feteanu, Pascale Picard, Chagri Boumaya, Jack Pollard, Colette Dib, Sukhvinder Sidhu, Laurent Debussche, Iris Valtingojer. Oncogenic HIPPO-YAP1: in vivo target validation of YAP1 in malignant mesothelioma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4858.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase belonging to the insulin receptor superfamily. Activating mutations in ALK are oncogenic and cause 10% to 15% of neuroblastoma cases. Similarly, activating ALK fusions have been detected in several cancers, including ALCL (NPM-ALK in 70% of patients) and NSCLC (EML4-ALK in 3% to 7% of patients). Several ALK inhibitors are currently undergoing clinical development, among which crizotinib has recently received marketing approval from the U.S. FDA under the name Xalkori. This nonselective ALK inhibitor has provided clinical benefit to lung cancer patients, but its sustained efficacy seems to be impaired by acquired drug resistance and several publications have already described ALK secondary mutations identified in patients who progressed while on crizotinib therapy. In this study, we have: Identified EML4-ALK mutations able to confer resistance to crizotinib. A screen using the Ba/F3 system found 24 residues in the ALK kinase domain where mutations can cause crizotinib resistance. Some of these mutations (L1152R, C1156Y, F1174L, G1202R, S1206F) have been identified in lung cancer patients progressing under crizotinib treatment Evaluated the activity of novel in-house and reference ALK kinase inhibitors against Ba/F3 cell lines stably expressing wild-type EML4-ALK and selected crizotinib-allele resistant mutantsPerformed in vivo head to head studies with a representative in-house ALK inhibitor and crizotinib to compare their pharmacodynamic properties This work resulted in the identification of an ALK kinase inhibitor with similar potency against wild-type EML4-ALK and crizotinib-allele resistant mutants and displaying significant target coverage in in vivo settings.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase belonging to the insulin receptor superfamily. Activating mutations in ALK are oncogenic and cause 10 to 15% of neuroblastoma cases. Similarly, activating ALK fusions have been detected in several cancers, including ALCL (NPM-ALK in 70% of patients) and NSCLC (EML4-ALK in 3–7% of patients). ALK inhibitors from different compound classes and kinase selectivity profiles are currently undergoing clinical development, among which crizotinib has recently received marketing approval from the US FDA. This non-selective ALK inhibitor has provided clinical benefit to lung cancer patients, but its sustained efficacy seems to be impaired by acquired drug resistance and several publications have already described ALK secondary mutations identified in patients who progressed while on crizotinib therapy. In this study, we have used the Ba/F3 system to identify EML4-ALK mutations able to confer resistance to crizotinib. To this end, we have generated a cDNA library containing random mutations in EML4-ALK using an E. Coli strain deficient in DNA repair pathways. The library was then subcloned in a retroviral vector and used to infect Ba/F3 cells. The cells were plated in 96 well microplates at an appropriate dilution and allowed to grow in the presence of 500 nM of crizotinib. The resistant cells were cultured and EML4-ALK was sequenced. Overall, this screen identified mutations at 24 positions in the ALK kinase domain. Some of these mutations (L1152R, C1156Y, F1174L) correspond to the ones known to cause resistance in lung cancer patients treated with crizotinib. Ba/F3 cell lines stably expressing a selected number of the 24 mutations have been generated and used to evaluate the activity of in-house and reference ALK kinase modulators. This work has shown that several mutations known to activate ALK in neuroblastoma are also able to confer resistance to crizotinib. The Ba/F3 cell lines expressing EML4-ALK mutants have been useful tools to characterize the activity profile of a range of ALK kinase inhibitors and several of our in-house compounds have been identified as active against the crizotinib-allele resistant mutants. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B76.