YAP Transcription Factors Research Articles

Cellular and Molecular Insights into the Divergence of Neural Stem Cells on Matrigel and Poly-l-lysine Interfaces.

Poly-l-lysine (PLL) and Matrigel, both classical coating materials for culture substrates in neural stem cell (NSC) research, present distinct interfaces whose effect on NSC behavior at cellular and molecular levels remains ambiguous. Our investigation reveals intriguing disparities: although both PLL and Matrigel interfaces are hydrophilic and feature amine functional groups, Matrigel stands out with lower stiffness and higher roughness. Based on this diversity, Matrigel surpasses PLL, driving NSC adhesion, migration, and proliferation. Intriguingly, PLL promotes NSC differentiation into astrocytes, whereas Matrigel favors neural differentiation and the physiological maturation of neurons. At the molecular level, Matrigel showcases a wider upregulation of genes linked to NSC behavior. Specifically, it enhances ECM-receptor interaction, activates the YAP transcription factor, and heightens glycerophospholipid metabolism, steering NSC proliferation and neural differentiation. Conversely, PLL upregulates genes associated with glial cell differentiation and amino acid metabolism and elevates various amino acid levels, potentially linked to its support for astrocyte differentiation. These distinct transcriptional and metabolic activities jointly shape the divergent NSC behavior on these substrates. This study significantly advances our understanding of substrate regulation on NSC behavior, offering novel insights into optimizing and targeting the application of these surface coating materials in NSC research.

Exosome-derived tRNA fragments tRF-GluCTC-0005 promotes pancreatic cancer liver metastasis by activating hepatic stellate cells

Early metastasis is the primary factor in the very poor prognosis of pancreatic ductal adenocarcinoma (PDAC), with liver metastasis being the most common form of distant metastasis in PDAC. To investigate the mechanism of PDAC liver metastasis, we found that PDAC cells can promote the formation of pre-metastatic niches (PMNs) through exosomes to facilitate liver metastasis in the early stage. In our study, hepatic stellate cells (HSCs) were treated with PDAC-derived exosomes (PDAC-exo), and the activation of HSCs was detected. A novel transfer RNA-derived fragment, the tRF-GluCTC-0005 was obtained by small RNA sequencing from serum exosomes of PDAC patients. Bioinformatics analysis and RNA pull-down assays revealed the interaction between WDR1 and tRF-GluCTC-0005. A KPC transgenic mouse model and an AAV-mediated sh-WDR1 mouse model were used to detect the mechanism of liver metastasis in vivo. Finally, the dual luciferase reporter assay, protein mutation truncation assay, Co-IP assay, and flow cytometry assay were used to explore the molecular mechanism in HSCs activation and PMNs formation. We found that the tRF-GluCTC-0005 in exosomes binds to the 3’ untranslated region of the mRNA of the WDRl in HSCs and increases mRNA stability. The N-terminals of WDR1 bind to the YAP protein directly, inhibit YAP phosphorylation, and promote the expression of YAP transcription factors. The tRF-GluCTC-0005 in PDAC-exo significantly recruits myeloid-derived suppressor cells (MDSCs) in the liver, creating a PMNs immunosuppressive microenvironment and further advancing liver metastasis from PDAC. Our results suggest that the key of PDAC liver metastasis is the activation of HSCs through upregulation of WDR1 by tRF-GluCTC-0005 in exosomes, which mediates the infiltration of MDSCs to form PMNs.

Abstract 2152: TNIK, a novel activator of FAK and YAP signaling, is a therapeutic target in lung squamous cell carcinoma

Abstract Lung squamous cell carcinoma (LSCC) is the second most common type of lung cancer. No precision therapies have been approved for the treatment of LSCC and the genetic drivers of LSCC are unknown in approximately 60% of the cases. As a result, treatment of LSCC is still limited to chemotherapy and/or radiotherapy. Analysis of LSCC samples from the TCGA reveals that nearly 50% of LSCC patients harbor distal amplification of the 3q chromosome that includes the resident protein kinase gene TNIK. Recent studies have pinpointed TNIK as a potential oncogenic driver in cancer cells with distal 3q amplification (Cancer Discov. 2013; 3:1044). However, the therapeutic potential of TNIK in LSCC remains unexplored. To determine if amplified TNIK is a genetic driver in LSCC, we analyzed TNIK expression in a panel of LSCC cell lines. We show that TNIK is overexpressed in LSCC cell lines in comparison with primary lung cells from healthy donors, consistent with observations in patient tumors based on analysis of TCGA data. Furthermore, we demonstrate that TNIK genetic depletion or pharmacological inhibition significantly reduces survival of LSCC cells with 3q amplification in vitro and in in vivo LSCC cell-line derived mouse xenografts. Importantly, we have generated LSCC patient-derived xenograft (PDX) mouse models to test TNIK inhibitors and have observed that inhibition of TNIK catalytic activity suppresses tumor growth in LSCC PDX mouse models. To elucidate the molecular mechanisms underpinning TNIK-driven LSCC cell viability we have used a combination of reverse-phase protein arrays, motif-driven screens for proteins with TNIK consensus phosphorylation sites, co-expression experiments, peptide mapping and mass spectrometry. Through these approaches, we have identified the tumor suppressor MERLIN as a novel TNIK substrate and determined that TNIK phosphorylates MERLIN at Ser13, Thr272 and Thr576. We also demonstrate that TNIK is required for stabilization of the YAP transcription factor, and activation of the focal adhesion kinase, FAK, two oncogenic pathways that are inhibited by MERLIN. In conclusion, we have pinpointed the protein kinase TNIK as a promising therapeutic target in LSCC that maintains survival of LSCC cells through modulation of novel a TNIK-MERLIN-YAP/FAK signaling pathway. Citation Format: Pedro Torres-Ayuso, Elvira An, Katherine M. Nyswaner, Daniel A. Ritt, Suzanne I. Specht, Sudipto Das, Thorkell Andresson, Christina M. Robinson, Simone Difilippantonio, Baktiar O. Karim, Benjamin E. Turk, Deborah K. Morrison, John Brognard. TNIK, a novel activator of FAK and YAP signaling, is a therapeutic target in lung squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2152.

3017 – MECHANISMS OF FLOW-DRIVEN TRANSCRIPTIONAL CONTROL OF HEMATOPOIETIC STEM CELL DEVELOPMENT VIA YAP/TAZ REGULATION

Hematopoietic stem cells (HSCs) are born from endothelial cells in the ventral dorsal aorta (VDA) that adopt hematopoietic cell fate during development. Physical forces of wall shear stress (WSS) and cyclic stretch (CS) produced by hemodynamic blood flow in the VDA are required to generate HSCs, but the mechanisms by which these forces are sensed and converted into a “stemness” regulatory program remain unknown. We recently showed that CS from blood flow stimulates activity of the YAP transcription factor (TF) in vitro in human induced pluripotent stem cell-derived CD34+ hemogenic endothelial cells (HE) and in vivo in zebrafish embryos to promote HSC production. YAP, and its paralogous TF TAZ/WWTR1, are members of the Hippo signaling pathway, initiate transcriptional responses downstream of mechanical stimuli, and require DNA binding cofactors to direct activation or repression of target genes. Via gene expression analysis and epistasis experiments modulating Notch signaling in zebrafish, we revealed YAP is responsible for maintenance, not initiation, of the hematopoietic program in newly specified HE. Notably, YAP loss-of-function mimics the phenotype of embryos deficient for the hematopoietic master regulator Runx1, with upregulation of the Notch ligand dll4. YAP/WWTR1 and RUNX TFs can physically interact in certain contexts. Surprisingly, using luciferase-based YAP- and RUNX-responsive reporter assays in HEK293 cells, we saw a potent synergistic effect of TAZ/RUNX1, but not YAP/RUNX1, in transcriptional regulation at RUNX binding elements. These findings position YAP signaling as a key transcriptional regulator of the endothelial-to-hematopoietic transition that produces HSCs from HE, and suggest a previously uninvestigated relationship between alternate utilization of the Hippo TFs and Runx1 that could underly the requirement of hemodynamic forces in this process. Hematopoietic stem cells (HSCs) are born from endothelial cells in the ventral dorsal aorta (VDA) that adopt hematopoietic cell fate during development. Physical forces of wall shear stress (WSS) and cyclic stretch (CS) produced by hemodynamic blood flow in the VDA are required to generate HSCs, but the mechanisms by which these forces are sensed and converted into a “stemness” regulatory program remain unknown. We recently showed that CS from blood flow stimulates activity of the YAP transcription factor (TF) in vitro in human induced pluripotent stem cell-derived CD34+ hemogenic endothelial cells (HE) and in vivo in zebrafish embryos to promote HSC production. YAP, and its paralogous TF TAZ/WWTR1, are members of the Hippo signaling pathway, initiate transcriptional responses downstream of mechanical stimuli, and require DNA binding cofactors to direct activation or repression of target genes. Via gene expression analysis and epistasis experiments modulating Notch signaling in zebrafish, we revealed YAP is responsible for maintenance, not initiation, of the hematopoietic program in newly specified HE. Notably, YAP loss-of-function mimics the phenotype of embryos deficient for the hematopoietic master regulator Runx1, with upregulation of the Notch ligand dll4. YAP/WWTR1 and RUNX TFs can physically interact in certain contexts. Surprisingly, using luciferase-based YAP- and RUNX-responsive reporter assays in HEK293 cells, we saw a potent synergistic effect of TAZ/RUNX1, but not YAP/RUNX1, in transcriptional regulation at RUNX binding elements. These findings position YAP signaling as a key transcriptional regulator of the endothelial-to-hematopoietic transition that produces HSCs from HE, and suggest a previously uninvestigated relationship between alternate utilization of the Hippo TFs and Runx1 that could underly the requirement of hemodynamic forces in this process.

TNIK, a novel activator of FAK and YAP signaling, is a therapeutic target in Lung Squamous Cell Carcinoma

Distal chromosome 3q amplification (3q26-29, also known as the 3q amplicon) is the most frequent genomic alteration in lung squamous cell carcinoma (LSCC). Analysis of LSCC samples from the TCGA reveals that nearly 50% of LSCC patients harbor the 3q amplicon that includes the resident protein kinase gene TNIK. Recent studies have pinpointed TNIK as a potential oncogenic driver in cancer cells with distal 3q amplification; however, the therapeutic potential of TNIK remains unexplored. We found that TNIK was highly expressed in LSCC cells with the 3q amplicon, while its expression was modest in cells that lacked the 3q amplicon, consistent with data from the TCGA. To evaluate TNIK as a target in LSCC, we generated stable, doxycycline-inducible cells expressing shRNA to deplete TNIK from LSCC cells and conducted functional assays to measure cell proliferation and survival. TNIK knockdown or inhibition of its kinase activity with a small molecule inhibitor significantly diminished the viability of LSCC cells with 3q amplification in vitro and in cell line-derived xenograft mouse models. We also observed that TNIK inhibitors significantly abrogated the growth of LSCC patient-derived xenografts and showed that TNIK inhibition induced apoptotic cell death in LSCC cells that harbor the 3q amplicon. Importantly, TNIK depletion or catalytic inhibition in LSCC cells that lack the 3q amplicon had no significant effect on cell survival. Finally, we used a combination of bioinformatics and proteomic analysis (RPPA, peptide mapping, and mass spectrometry) to define the underlying mechanisms driving TNIK mediated cancer cell survival. We identified the tumor suppressor MERLIN as a novel TNIK substrate and determined that TNIK phosphorylates MERLIN at serine 13 and 315. We also show that TNIK is required to maintain FAK activation and stabilize the YAP transcription factor, two oncogenic pathways inhibited by MERLIN. In conclusion, our results demonstrate that TNIK maintains survival of LSCC cells through modulation of a novel TNIK-MERLIN-YAP/FAK signaling pathway and validate TNIK inhibitors in pre-clinical models of LSCC, including patient-derived xenografts. In summary, we have pinpointed the protein kinase TNIK as a promising therapeutic target for the treatment of LSCC patients with distal chromosome 3q amplification.

3039 – YAP REGULATES HEMATOPOIETIC STEM CELL FORMATION IN RESPONSE TO THE BIOMECHANICAL FORCES OF BLOOD FLOW

Hematopoietic stem cells (HSCs) are born from cells in the dorsal aorta that adopt hematopoietic potential and commitment during development. Physical forces, namely wall shear stress (WSS) and cyclic stretch (CS), produced by hemodynamic blood flow are required to generate HSCs from arterial endothelium, but the mechanisms by which these forces are transduced into a "stemness" regulatory program remain unknown. Using a novel "dorsal-aorta-on-a-chip" microfluidic platform, we demonstrate that exposure to WSS and CS increases expression of the hematopoietic transcription factor RUNX1 in human CD34+ hemogenic endothelial cells derived from induced pluripotent stem cells. Notably, CS specifically induced activity of the YAP transcription factor, a mechanically-activated regulator of organ size and pluripotency. We corroborate these findings in a zebrafish model, and demonstrate that loss or gain of YAP function in vivo can blunt or augment the production of HSCs during definitive hematopoiesis. Importantly, we find that YAP is responsible for the maintenance, not initiation, of the hematopoietic program in newly specified hemogenic endothelial cells. Molecularly, we identify a stretch-induced, RhoGTPase-dependent mechanotransduction network controlling YAP nuclear availability to regulate RUNX1 expression, both in zebrafish and human cells. Moreover, we show that small molecule stimulation of RhoGTPases can rescue HSC production in zebrafish embryos with no flow, and enhance the hematopoietic potential of human iPSC-derived CD34+ endothelial cells grown in static culture. Together these findings uncover YAP as a transcriptional regulator of stem cell fate commitment during tissue morphogenesis, and suggest a pharmacologically amenable mechanotransduction pathway that could be exploited to improve the in vitro derivation of HSCs from human cells for therapeutic purposes.

Culture Into Perfusion-Assisted Bioreactor Promotes Valve-Like Tissue Maturation of Recellularized Pericardial Membrane.

Derivation of tissue-engineered valve replacements is a strategy to overcome the limitations of the current valve prostheses, mechanical, or biological. In an effort to set living pericardial material for aortic valve reconstruction, we have previously assessed the efficiency of a recellularization strategy based on a perfusion system enabling mass transport and homogenous distribution of aortic valve-derived “interstitial” cells inside decellularized pericardial material. In the present report, we show that alternate perfusion promoted a rapid growth of valve cells inside the pericardial material and the activity of a proliferation-supporting pathway, likely controlled by the YAP transcription factor, a crucial component of the Hippo-dependent signaling cascade, especially between 3 and 14 days of culture. Quantitative mass spectrometry analysis of protein content in the tissue constructs showed deposition of valve proteins in the decellularized pericardium with a high variability at day 14 and a reproducible tissue maturation at 21 days. These results represent a step forward in the definition of strategies to produce a fully engineered tissue for replacing the calcified leaflets of failing aortic valves.

Boolean model of anchorage dependence and contact inhibition points to coordinated inhibition but semi-independent induction of proliferation and migration

Epithelial cells respond to their physical neighborhood with mechano-sensitive behaviors required for development and tissue maintenance. These include anchorage dependence, matrix stiffness-dependent proliferation, contact inhibition of proliferation and migration, and collective migration that balances cell crawling with the maintenance of cell junctions. While required for development and tissue repair, these coordinated responses to the microenvironment also contribute to cancer metastasis. Predictive models of the signaling networks that coordinate these behaviors are critical in controlling cell behavior to halt disease. Here we propose a Boolean regulatory network model that synthesizes mechanosensitive signaling that links anchorage to a matrix of varying stiffness and cell density sensing to contact inhibition, proliferation, migration, and apoptosis. Our model can reproduce anchorage dependence and anoikis, detachment-induced cytokinesis errors, the effect of matrix stiffness on proliferation, and contact inhibition of proliferation and migration by two mechanisms that converge on the YAP transcription factor. In addition, we offer testable predictions related to cell cycle-dependent anoikis sensitivity, the molecular requirements for abolishing contact inhibition, and substrate stiffness dependent expression of the catalytic subunit of PI3K. Moreover, our model predicts heterogeneity in migratory vs. non-migratory phenotypes in sub-confluent monolayers, and co-inhibition but semi-independent induction of proliferation vs. migration as a function of cell density and mitogenic stimulation. Our model serves as a stepping-stone towards modeling mechanosensitive routes to the epithelial to mesenchymal transition, capturing the effects of the mesenchymal state on anoikis resistance, and understanding the balance between migration versus proliferation at each stage of the epithelial to mesenchymal transition.

P5382Geometry and strain sensing dictate YAP-dependent evolution of human cardiac stromal cells toward myofibroblasts in the cardiosphere organoid model

Abstract Background Cardiac stromal cells are the most abundant cell type present in the heart, where they have an important homeostatic function in matrix renewal and electromechanical coupling. We have shown that YAP transcription factor (a component of the Hippo pathway) is important for the proliferation of these cells and, potentially, pathologic cardiac matrix stiffening in heart failure. Purpose Given the role of Hippo pathway in cell mechanosensation, we assessed the conditions for activation of the YAP-dependent transcriptional pathway related to geometry/strain sensing using human “cardiosphere”-derived cells. Methods Human cardiospheres (C-Sp) and C-Sp-derived cells (CDCs) were obtained from fragments of atrial appendages of patients admitted for AoC bypass. C-Sps and CDCs were cultured using published methods. To analyze cell/nuclear geometry in 2D/3D culture, we employed an adapting thresholding algorithm allowing segmentation of cell membrane/nuclei shape and the relative abundance of nuclear/cytoplasmic fluorescence intensities from z-stacked confocal immunofluorescence images. Inhibitors of signaling cascades converging onto actin cytoskeleton polymerization or of YAP/TEADs interaction were used to assess YAP target genes transcription. Results Previous results showed stiffness sensitivity of YAP nuclear translocation process. Namely, CDCs plated onto stiff (E >50kPa) culture plates exhibited high ratios of nuclear/cytoplasmic YAP, as compared to cells plated on substrates with lower stiffness (E ∼ 10kPa). Analogously, cells with a stretched cytoplasm/nuclear shape in the border regions of the 3D organoids exhibited a higher YAP nuclear localization level and expression of fibrotic markers compared with cells with a more round geometry in the center of the spheres. To uncouple the cytoskeleton tensioning-dependent YAP nuclear shuttling, we treated CDCs plated onto stiff substrates with blebbistatin and Y27632. This showed a fully reversible YAP relocation from the nucleus to the cytoplasm and a reversion of canonical target genes (CYR61, ANKRD1 and CTGF) expression. In parallel, a “softening” process of the nucleus, likely due to release from cytoskeleton traction forces, was observed by confocal imaging along the cellular z-axis. Interestingly, use of Verteporfin, a drug directly interfering with the interaction of YAP/TAZ complex with TEADs DNA binding proteins showed reversion of pro-fibrotic YAP targets independent of high cytoskeleton tensioning. Conclusions These results establish, for the first time, a deterministic relationship between pro-pathologic evolution of human adult cardiac stromal cells and activation of mechanosensing-dependent pathways. They also provide quantitative criteria for interpreting fibrotic evolution of the heart based on modifications of myocardial architecture and mechanical compliance, as well as the ability of the cells to generate tension forces affecting the nuclear structure.

Yeast AP-1 like transcription factors (Yap) and stress response: a current overview.

Yeast adaptation to stress has been extensively studied. It involves large reprogramming of genome expression operated by many, more or less specific, transcription factors. Here, we review our current knowledge on the function of the eight Yap transcription factors (Yap1 to Yap8) in Saccharomyces cerevisiae, which were shown to be involved in various stress responses. More precisely, Yap1 is activated under oxidative stress, Yap2/Cad1 under cadmium, Yap4/Cin5 and Yap6 under osmotic shock, Yap5 under iron overload and Yap8/Arr1 by arsenic compounds. Yap3 and Yap7 seem to be involved in hydroquinone and nitrosative stresses, respectively. The data presented in this article illustrate how much knowledge on the function of these Yap transcription factors is advanced. The evolution of the Yap family and its roles in various pathogenic and non-pathogenic fungal species is discussed in the last section.

Abstract 475: Human Saphenous Vein Progenitor Cells Are Susceptible to Mechanical Stimulation. Novel Insights in Pathologic Programming of Saphenous Vein Bypass Graft Disease

Background: Implantation of saphenous vein (SV) grafts into coronary position determines structural vessel wall remodeling and intimal hyperplasia. The role of the altered wall mechanics and cell-based mechanosensing has been recently implicated in the priming of this pathologic process. We investigated the effects of cyclic uniaxial strain on human saphenous vein progenitor cells (SVPs), a cell type endowed with pericyte stem cell characteristics resident in the adventitia. Methods: CD34 + CD31 - SVPs were isolated with immuno-magnetic sorting (MACS) from SVs of patients (age 58±12.6, Mean±SD) undergoing saphenectomy. Cells were subject to uniaxial strain (10% elongation; 1Hz) for 24 and 72 hrs using the FlexCell system. Cell orientation, immunofluorescence and western analyses were performed to assess the effects of strain on cell orientation/shape, cell cycle and activity of the YAP/Hippo-dependent mechanotransduction machinery. RNA-sequencing from control vs. strained SVPs was performed at both time points using RNA from cells of 5 independent donors. Results: Results indicated an increase in the expression of the cell cycle-associated markers Ki67 and pHH3 in mechanically stimulated vs. control SVPs at 24 hrs, followed by a significant reduction at 72 hrs of stimulation. Variations in cell shape was observed as verified by a significant change in the nuclei orientation in the strain field as well as in the cell shape index/spread areas at both time points. Immunofluorescence revealed a significant increase in cells showing a nuclear localization of the YAP transcription factor at 72hrs. In line with these findings Western analyses indicated a significant decrease in the ratio between phosphorylated/total YAP and its upstream kinase LATS in mechanically stimulated vs. control SVPs, suggesting an inhibition of the HIPPO kinase pathway by mechanosensing. RNASeq gene expression analyses showed a coherent modification of gene expression pathways and the upregulation of a specific HIPPO/YAP/TEAD gene expression signature in SVPs mechanically stimulated for 72hrs. Conclusions: These findings demonstrate the direct susceptibility of human SVPs to pathologic strain and identifies this cell population as mechano-perceptors in the vein wall.

Wnt activated β-catenin and YAP proteins enhance the expression of non-coding RNA component of RNase MRP in colon cancer cells.

RMRP, the RNA component of mitochondrial RNA processing endoribonuclease, is a non-coding RNA (ncRNA) part of the RNase MRP complex functioning in mitochondrial and ribosomal RNA processing. Even though various mutations in the RMRP gene are linked to developmental defects and pathogenesis, its relevance to cancer etiology has not been well established. Here we examined the expression of RMRP and found a significant increase in colorectal and breast cancer patient tissues. So we tested whether the oncogenic signaling pathways, Wnt/β-catenin and Hippo/YAP pathways, are relevant to the enhanced expression of RMRP in cancer cells because of the predicted β-catenin/TCF and YAP/TBX5 elements in the upstream regions of the RMRP gene. As expected, Wnt signal activation significantly induced the RMRP transcription thru β-catenin and YAP transcription factors. More importantly, YAP protein was critical for RMRP transcription by association to the proximal site near the transcription start site of the RMRP gene, a Pol III promoter, along with β-catenin and TBX5 proteins. We propose that the interplay of Wnt and Hippo signaling pathways could regulate target genes, coding or non-coding, by the β-catenin/YAP/TBX5 transcription complex in cancer cells.

Abstract 2200: A rational approach for discovery of inhibitors of YAP-TEAD interaction

Abstract The Hippo signalling pathway plays a major role in organ growth regulation and tumorigenesis. By sensing contact inhibition, the central kinase network of the hippo pathway controls nuclear translocation and activity of the YAP transcription factor. Once translocated in the nucleus, YAP interacts with the TEAD family of transcription factors and drives expression of gene involved in cell survival and proliferation. Deregulation of the hippo pathway frequently occurs in cancers and high nuclear expression of YAP has been documented in many tumors including lung, colorectal, ovarian and skin cancers. These data suggest that targeting the hippo pathway is a new therapeutic option for the treatment of a large number of cancers. Several approaches are currently proposed to target the hippo pathway such as inhibition of kinases and GPCRs that are upstream regulators of YAP nuclear translocation. However, because YAP and TEAD are the downstream effectors of the Hippo pathway, we believe that targeting YAP-TEAD interaction is the most promising approach to selectively counteract abnormal activity of the Hippo pathway. We have started a drug discovery program aimed at identifying potent inhibitors of YAP-TEAD interaction, based on a combined FBLD/HTS strategy. Druggability of the TEAD protein was assessed by a fragment screen using NMR and SPR technologies. This approach, conforted by assigned HSQC protein NMR information, provided fragment hits which are used as starting points for the identification of more potent binders. We have also designed a dedicated AlphaScreen assay to validate the ability of our compounds to disrupt the YAP-TEAD interaction. Fifty thousand compounds of the Inventiva's library were screened using the AlphaScreen. Several hits in the micromolar range were identified and further confirmed using SPR. Hits are currently tested for their ability to disrupt YAP-TEAD interaction in a cellular context with a transactivation assay and for their ability to block tumoral cell proliferation. Citation Format: Claudia Fromond, Laurent Chene, Anne Soude, Martine Barth, Christian Montalbetti, Pierre Broqua. A rational approach for discovery of inhibitors of YAP-TEAD interaction. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2200. doi:10.1158/1538-7445.AM2015-2200

G.P.14: From muscular architecture to function: The involvement of FAT1 protocadherin in FSHD

Facio Scapulo Humeral Dystrophy (FSHD) is a muscle disease that asymmetrically affects flat muscles. Non-muscle symptoms are deafness and retinopathies. Defects very evocative of FSHD have been observed in mouse model partially deficient for FAT1. In addition, genetic screening of neuromuscular disease patients enabled us to identify the FAT1 mutations in FSHD-like patients. The family of FAT protocadherins (FAT 1–4) is well known to be involved in the Wnt/PCP and in the Hippo pathway in Drosophila, while it plays key roles in homeostasis of mammalian tissues. FAT1 expression is localized in developing limb buds, somites and neural tube as well as in adult kidney. Based on these data and on our previous results, we postulated that FAT1 plays a role in generation and homeostasis of flat muscle tissues. To understand which proteins compose the transduction pathway led by FAT1 in mammals, we first looked at the stability and activation of YAP transcription factor in the Hippo pathway. Although preliminary, results confirmed that FAT1 overexpression affects YAP/TAZ phosphorylation, nuclear localization and transcriptional activity, which may stop cell proliferation and affect cell polarity. Indeed, localization of YAP/TAZ depends from elasticity of the cellular environment through signal transduction from the extracellular matrix (ECM). In mesenchymal stem cells, TAZ and YAP are cytoplasmic when cells are on flexible substrates while nuclear on rigid matrices. Therefore, we plan to test muscle cells reaction to different levels of matrix rigidity and to measure functional consequences of FAT1 alterations to these mechanical stresses. In perspective, our observations will help explain the molecular mechanism involved in the differentiation and tissue response to cellular environment. Moreover, FAT1 alteration may lead to modification of ECM stiffness perception during flat muscle fibers development, thus contributing to pathological mechanism of FSHD.

ZO Proteins Redundantly Regulate the Transcription Factor DbpA/ZONAB

The localization and activities of DbpA/ZONAB and YAP transcription factors are in part regulated by the density-dependent assembly of epithelial junctions. DbpA activity and cell proliferation are inhibited by exogenous overexpression of the tight junction (TJ) protein ZO-1, leading to a model whereby ZO-1 acts by sequestering DbpA at the TJ. However, mammary epithelial cells and mouse tissues knock-out for ZO-1 do not show increased proliferation, as predicted by this model. To address this discrepancy, we examined the localization and activity of DbpA and YAP in Madin-Darby canine kidney cells depleted either of ZO-1, or one of the related proteins ZO-2 and ZO-3 (ZO proteins), or all three together. Depletion of only one ZO protein had no effect on DbpA localization and activity, whereas depletion of ZO-1 and ZO-2, which is associated with reduced ZO-3 expression, resulted in increased DbpA localization in the cytoplasm. Only depletion of ZO-2 reduced the nuclear import of YAP. Mammary epithelial (Eph4) cells KO for ZO-1 showed junctional DbpA, demonstrating that ZO-1 is not required to sequester DbpA at junctions. However, further depletion of ZO-2 in Eph4 ZO-1KO cells, which do not express ZO-3, caused decreased junctional localization and expression of DbpA, which were rescued by the proteasome inhibitor MG132. In vitro binding assays showed that full-length ZO-1 does not interact with DbpA. These results show that ZO-2 is implicated in regulating the nuclear shuttling of YAP, whereas ZO proteins redundantly control the junctional retention and stability of DbpA, without affecting its shuttling to the nucleus.

Mechanotransduction and YAP-dependent matrix remodelling is required for the generation and maintenance of cancer-associated fibroblasts

To learn more about cancer-associated fibroblasts (CAFs), we have isolated fibroblasts from different stages of breast cancer progression and analysed their function and gene expression. These analyses reveal that activation of the YAP transcription factor is a signature feature of CAFs. YAP function is required for CAFs to promote matrix stiffening, cancer cell invasion and angiogenesis. Remodelling of the ECM and promotion of cancer cell invasion requires the actomyosin cytoskeleton. YAP regulates the expression of several cytoskeletal regulators, including ANLN, and DIAPH3, and controls the protein levels of MYL9/MLC2. Matrix stiffening further enhances YAP activation, thus establishing a feed-forward self-reinforcing loop that helps to maintain the CAF phenotype. Actomyosin contractility and Src function are required for YAP activation by stiff matrices. Further, transient ROCK inhibition is able to disrupt the feed-forward loop leading to a long-lasting reversion of the CAF phenotype.

The Yap family and its role in stress response

The budding yeast Saccharomyces cerevisiae possesses a very flexible and complex programme of gene expression when exposed to several environmental challenges. Homeostasis is achieved through a highly coordinated mechanism of transcription regulation involving several transcription factors, each one acting singly or in combination to perform specific functions. Here, we review our current knowledge of the function of the Yap transcription factors in stress response. They belong to b-ZIP proteins comprising eight members with specificity at the DNA-binding domain distinct from that of the conventional yeast AP-1 factor, Gcn4. We finish with new insights into the links of transcriptional networks controlling several cellular processes. The data reviewed in this article illustrate how much our comprehension of the biology of Yap family involved in stress response has advanced, and how much research is still needed to unravel the complexity of the role of these transcriptional factors. The complexities of these regulatory interactions, as well as the dynamics of these processes, are important to understand in order to elucidate the control of stress response, a highly conserved process in eukaryotes.