Transcription factors TEAD2 and E2A globally repress acetyl-CoA synthesis to promote tumorigenesis.

Acetyl-coenzyme A (acetyl-CoA) plays an important role in metabolism, gene expression, signaling, and other cellular processes via transfer of its acetyl group to proteins and metabolites. However, the synthesis and usage of acetyl-CoA in disease states such as cancer are poorly characterized. Here, we investigated global acetyl-CoA synthesis and protein acetylation in a mouse model and patient samples of hepatocellular carcinoma (HCC). Unexpectedly, we found that acetyl-CoA levels are decreased in HCC due to transcriptional downregulation of all six acetyl-CoA biosynthesis pathways. This led to hypo-acetylation specifically of non-histone proteins, including many enzymes in metabolic pathways. Importantly, repression of acetyl-CoA synthesis promoted oncogenic dedifferentiation and proliferation. Mechanistically, acetyl-CoA synthesis was repressed by the transcription factors TEAD2 and E2A, previously unknown to control acetyl-CoA synthesis. Knockdown of TEAD2 and E2A restored acetyl-CoA levels and inhibited tumor growth. Our findings causally link transcriptional reprogramming of acetyl-CoA metabolism, dedifferentiation, and cancer.

YAP-VGLL4 antagonism defines the major physiological function of the Hippo signaling effector YAP.

The Hippo-YAP signaling pathway plays a critical role in development, homeostasis, regeneration, and tumorigenesis by converging on YAP, a coactivator for the TEAD family DNA-binding transcription factors, to regulate downstream transcription programs. Given its pivotal role as the nuclear effector of the Hippo pathway, YAP is indispensable in multiple developmental and tissue contexts. Here we report that the essentiality of YAP in liver and lung development can be genetically bypassed by simultaneous inactivation of the TEAD corepressor VGLL4. This striking antagonistic epistasis suggests that the major physiological function of YAP is to antagonize VGLL4. We further show that the YAP-VGLL4 antagonism plays a widespread role in regulating Hippo pathway output beyond normal development, as inactivation of Vgll4 dramatically enhanced intrahepatic cholangiocarcinoma formation in Nf2-deficient livers and ameliorated CCl4-induced damage in normal livers. Interestingly, Vgll4 expression is temporally regulated in development and regeneration and, in certain contexts, provides a better indication of overall Hippo pathway output than YAP phosphorylation. Together, these findings highlight the central importance of VGLL4-mediated transcriptional repression in Hippo pathway regulation and inform potential strategies to modulate Hippo signaling in cancer and regenerative medicine.

Increased ACTL6A occupancy within mSWI/SNF chromatin remodelers drives human squamous cell carcinoma.

Mammalian SWI/SNF (BAF) chromatin remodelers play dosage-sensitive roles in many human malignancies and neurologic disorders. The gene encoding the BAF subunit actin-like 6a (ACTL6A) is amplified early in the development of many squamous cell carcinomas (SCCs), but its oncogenic role remains unclear. Here we demonstrate that ACTL6A overexpression leads to its stoichiometric assembly into BAF complexes and drives their interaction and engagement with specific regulatory regions in the genome. In normal epithelial cells, ACTL6A was substoichiometric to other BAF subunits. However, increased ACTL6A levels by ectopic expression or in SCC cells led to near saturation of ACTL6A within BAF complexes. Increased ACTL6A occupancy enhanced polycomb opposition genome-wide to activate SCC genes and facilitated the co-dependent loading of BAF and TEAD-YAP complexes on chromatin. Both mechanisms appeared to be critical and function as a molecular AND gate for SCC initiation and maintenance, thereby explaining the specificity of the role of ACTL6A amplification in SCCs.

TEAD2 initiates ground-state pluripotency by mediating chromatin looping.

The transition of mouse embryonic stem cells (ESCs) between serum/LIF and 2i(MEK and GSK3 kinase inhibitor)/LIF culture conditions serves as a valuable model for exploring the mechanisms underlying ground and confused pluripotent states. Regulatory networks comprising core and ancillary pluripotency factors drive the gene expression programs defining stable naïve pluripotency. In our study, we systematically screened factors essential for ESC pluripotency, identifying TEAD2 as an ancillary factor maintaining ground-state pluripotency in 2i/LIF ESCs and facilitating the transition from serum/LIF to 2i/LIF ESCs. TEAD2 exhibits increased binding to chromatin in 2i/LIF ESCs, targeting active chromatin regions to regulate the expression of 2i-specific genes. In addition, TEAD2 facilitates the expression of 2i-specific genes by mediating enhancer-promoter interactions during the serum/LIF to 2i/LIF transition. Notably, deletion of Tead2 results in reduction of a specific set of enhancer-promoter interactions without significantly affecting binding of chromatin architecture proteins, CCCTC-binding factor (CTCF), and Yin Yang 1 (YY1). In summary, our findings highlight a novel prominent role of TEAD2 in orchestrating higher-order chromatin structures of 2i-specific genes to sustain ground-state pluripotency.

Comparison of tumor-associated YAP1 fusions identifies a recurrent set of functions critical for oncogenesis.

YAP1 is a transcriptional coactivator and the principal effector of the Hippo signaling pathway, which is causally implicated in human cancer. Several YAP1 gene fusions have been identified in various human cancers and identifying the essential components of this family of gene fusions has significant therapeutic value. Here, we show that the YAP1 gene fusions YAP1-MAMLD1, YAP1-FAM118B, YAP1-TFE3, and YAP1-SS18 are oncogenic in mice. Using reporter assays, RNA-seq, ChIP-seq, and loss-of-function mutations, we can show that all of these YAP1 fusion proteins exert TEAD-dependent YAP activity, while some also exert activity of the C'-terminal fusion partner. The YAP activity of the different YAP1 fusions is resistant to negative Hippo pathway regulation due to constitutive nuclear localization and resistance to degradation of the YAP1 fusion proteins. Genetic disruption of the TEAD-binding domain of these oncogenic YAP1 fusions is sufficient to inhibit tumor formation in vivo, while pharmacological inhibition of the YAP1-TEAD interaction inhibits the growth of YAP1 fusion-expressing cell lines in vitro. These results highlight TEAD-dependent YAP activity found in these gene fusions as critical for oncogenesis and implicate these YAP functions as potential therapeutic targets in YAP1 fusion-positive tumors.

Therapeutic targeting of TEAD transcription factors in cancer.

The Hippo signaling pathway inhibits the activity of the oncogenic YAP (Yes-associated protein)/TAZ (transcriptional co-activator with PDZ-binding motif)-TEAD (TEA/ATTS domain) transcriptional complex. In cancers, inactivating mutations in upstream Hippo components and/or enhanced activity of YAP/TAZ and TEAD have been observed. The activity of this transcriptional complex can be effectively inhibited by targeting the TEAD family of transcription factors. The development of TEAD inhibitors has been driven by the discovery that TEAD has druggable hydrophobic pockets, and is currently at the clinical development stage. Three small molecule TEAD inhibitors are currently being tested in Phase I clinical trials. In this review, we highlight the role of TEADs in cancer, discuss various avenues through which TEAD activity can be inhibited, and outline the opportunities for the administration of TEAD inhibitors.

YAP antagonizes TEAD-mediated AR signaling and prostate cancer growth.

Hippo signaling restricts tumor growth by inhibiting the oncogenic potential of YAP/TAZ-TEAD transcriptional complex. Here, we uncover a context-dependent tumor suppressor function of YAP in androgen receptor (AR) positive prostate cancer (PCa) and show that YAP impedes AR+ PCa growth by antagonizing TEAD-mediated AR signaling. TEAD forms a complex with AR to enhance its promoter/enhancer occupancy and transcriptional activity. YAP and AR compete for TEAD binding and consequently, elevated YAP in the nucleus disrupts AR-TEAD interaction and prevents TEAD from promoting AR signaling. Pharmacological inhibition of MST1/2 or LATS1/2, or transgenic activation of YAP suppressed the growth of PCa expressing therapy resistant AR splicing variants. Our study uncovers an unanticipated crosstalk between Hippo and AR signaling pathways, reveals an antagonistic relationship between YAP and TEAD in AR+ PCa, and suggests that targeting the Hippo signaling pathway may provide a therapeutical opportunity to treat PCa driven by therapy resistant AR variants.

A Non-canonical Role of YAP/TEAD Is Required for Activation of Estrogen-Regulated Enhancers in Breast Cancer.

YAP/TEAD are nuclear effectors of the Hippo pathway, regulating organ size and tumorigenesis largely through promoter-associated function. However, their function as enhancer regulators remains poorly understood. Through an in vivo proximity-dependent labeling (BioID) technique, we identified YAP1 and TEAD4 protein as co-regulators of ERα on enhancers. The binding of YAP1/TEAD4 to ERα-bound enhancers is augmented upon E2 stimulation and is required for the induction of E2/ERα target genes and E2-induced oncogenic cell growth. Furthermore, their enhancer binding is a prerequisite for enhancer activation marked by eRNA transcription and for the recruitment of the enhancer activation machinery component MED1. The binding of TEAD4 on active ERE-containing enhancers is independent of its DNA-binding behavior, and instead, occurs through protein-tethering trans-binding. Our data reveal a non-canonical function of YAP1 and TEAD4 as ERα cofactors in regulating cancer growth, highlighting the potential of YAP/TEAD as possible actionable drug targets for ERα+ breast cancer.

Identification of resistance mechanisms to small-molecule inhibition of TEAD-regulated transcription.

The Hippo tumor suppressor pathway controls transcription by regulating nuclear abundance of YAP and TAZ, which activate transcription with the TEAD1-TEAD4 DNA-binding proteins. Recently, several small-molecule inhibitors of YAP and TEADs have been reported, with some entering clinical trials for different cancers with Hippo pathway deregulation, most notably, mesothelioma. Using genome-wide CRISPR/Cas9 screens we reveal that mutations in genes from the Hippo, MAPK, and JAK-STAT signaling pathways all modulate the response of mesothelioma cell lines to TEAD palmitoylation inhibitors. By exploring gene expression programs of mutant cells, we find that MAPK pathway hyperactivation confers resistance to TEAD inhibition by reinstating expression of a subset of YAP/TAZ target genes. Consistent with this, combined inhibition of TEAD and the MAPK kinase MEK, synergistically blocks proliferation of multiple mesothelioma and lung cancer cell lines and more potently reduces the growth of patient-derived lung cancer xenografts in vivo. Collectively, we reveal mechanisms by which cells can overcome small-molecule inhibition of TEAD palmitoylation and potential strategies to enhance the anti-tumor activity of emerging Hippo pathway targeted therapies.

YAP and TAZ Are Not Identical Twins.

Yes-associated protein (YAP) and TAZ (WW domain containing transcription regulator 1, or WWTR1) are paralog transcriptional regulators, able to integrate mechanical, metabolic, and signaling inputs to regulate cell growth and differentiation during development and neoplastic progression. YAP and TAZ hold common and distinctive structural features, reflecting only partially overlapping regulatory mechanisms. The two paralogs interact with both shared and specific transcriptional partners and control nonidentical transcriptional programs. Although most of the available literature considers YAP and TAZ as functionally redundant, they play distinctive or even contrasting roles in different contexts. The issue of their divergent roles is currently underexplored but holds fundamental implications for mechanistic and translational studies. Here, we aim to review the available literature on the biological functions of YAP and TAZ, highlighting differential roles that distinguish these two paralogues.

The tumor suppressor NF2 modulates TEAD4 stability and activity in Hippo signaling via direct interaction.

As an output effector of the Hippo signaling pathway, the TEAD transcription factor and co-activator YAP play crucial functions in promoting cell proliferation and organ size. The tumor suppressor NF2 has been shown to activate LATS1/2 kinases and interplay with the Hippo pathway to suppress the YAP-TEAD complex. However, whether and how NF2 could directly regulate TEAD remains unknown. We identified a direct link and physical interaction between NF2 and TEAD4. NF2 interacted with TEAD4 through its FERM domain and C-terminal tail and decreased the protein stability of TEAD4 independently of LATS1/2 and YAP. Furthermore, NF2 inhibited TEAD4 palmitoylation and induced the cytoplasmic translocation of TEAD4, resulting in ubiquitination and dysfunction of TEAD4. Moreover, the interaction with TEAD4 is required for NF2 function to suppress cell proliferation. These findings reveal an unanticipated role of NF2 as a binding partner and inhibitor of the transcription factor TEAD, shedding light on an alternative mechanism of how NF2 functions as a tumor suppressor through the Hippo signaling cascade.

Upregulation of CYR61 by TGF-ß and YAP signaling exerts a counter-suppression of hepatocellular carcinoma.

Transforming growth factor-ß (TGF-ß) and Hippo signaling are two critical pathways engaged in cancer progression by regulating both oncogenes and tumor suppressors, yet how the two pathways coordinately exert their functions in the development of hepatocellular carcinoma (HCC) remains elusive. In this study, we firstly conducted an integrated analysis of public liver cancer databases and our experimental TGF-ß target genes, identifying CYR61 as a pivotal candidate gene relating to HCC development. The expression of CYR61 is downregulated in clinical HCC tissues and cell lines than that in the normal counterparts. Evidence revealed that CYR61 is a direct target gene of TGF-ß in liver cancer cells. In addition, TGF-ß-stimulated Smad2/3 and the Hippo pathway downstream effectors YAP and TEAD4 can form a protein complex on the promoter of CYR61, thereby activating the promoter activity and stimulating CYR61 gene transcription in a collaborative manner. Functionally, depletion of CYR61 enhanced TGF-ß- or YAP-mediated growth and migration of liver cancer cells. Consistently, ectopic expression of CYR61 was capable of impeding TGF-ß- or YAP-induced malignant transformation of HCC cells in vitro and attenuating HCC xenograft growth in nude mice. Finally, transcriptomic analysis indicates that CYR61 can elicit an antitumor program in liver cancer cells. Together, these results add new evidence for the crosstalk between TGF-ß and Hippo signaling and unveil an important tumor suppressor function of CYR61 in liver cancer.

Estrogen receptor activation remodels TEAD1 gene expression to alleviate hepatic steatosis.

Sex-based differences in obesity-related hepatic malignancies suggest the protective roles of estrogen. Using a preclinical model, we dissected estrogen receptor (ER) isoform-driven molecular responses in high-fat diet (HFD)-induced liver diseases of male and female mice treated with or without an estrogen agonist by integrating liver multi-omics data. We found that selective ER activation recovers HFD-induced molecular and physiological liver phenotypes. HFD and systemic ER activation altered core liver pathways, beyond lipid metabolism, that are consistent between mice and primates. By including patient cohort data, we uncovered that ER-regulated enhancers govern central regulatory and metabolic genes with clinical significance in metabolic dysfunction-associated steatotic liver disease (MASLD) patients, including the transcription factor TEAD1. TEAD1 expression increased in MASLD patients, and its downregulation by short interfering RNA reduced intracellular lipid content. Subsequent TEAD small molecule inhibition improved steatosis in primary human hepatocyte spheroids by suppressing lipogenic pathways. Thus, TEAD1 emerged as a new therapeutic candidate whose inhibition ameliorates hepatic steatosis.

TEAD Proteins Associate With DNA Repair Proteins to Facilitate Cellular Recovery From DNA Damage.

Transcriptional enhanced associate domain family members 1 to 4 (TEADs) are a family of four transcription factors and the major transcriptional effectors of the Hippo pathway. In order to activate transcription, TEADs rely on interactions with other proteins, such as the transcriptional effectors Yes-associated protein and transcriptional co-activator with PDZ-binding motif. Nuclear protein interactions involving TEADs influence the transcriptional regulation of genes involved in cell growth, tissue homeostasis, and tumorigenesis. Clearly, protein interactions for TEADs are functionally important, but the full repertoire of TEAD interaction partners remains unknown. Here, we employed an affinity purification mass spectrometry approach to identify nuclear interacting partners of TEADs. We performed affinity purification mass spectrometry experiment in parallel in two different cell types and compared a wildtype TEAD bait protein to a nuclear localization sequence mutant that does not localize to the nucleus. We quantified the results using SAINT analysis and found a significant enrichment of proteins linked to DNA damage including X-ray repair cross-complementing protein 5 (XRCC5), X-ray repair cross-complementing protein 6 (XRCC6), poly(ADP-ribose) polymerase 1 (PARP1), and Rap1-interacting factor 1 (RIF1). In cellular assays, we found that TEADs co-localize with DNA damage-induced nuclear foci marked by histone H2AX phosphorylated on S139 (γH2AX) and Rap1-interacting factor 1. We also found that depletion of TEAD proteins makes cells more susceptible to DNA damage by various agents and that depletion of TEADs promotes genomic instability. Additionally, depleting TEADs dampens the efficiency of DNA double-stranded break repair in reporter assays. Our results connect TEADs to DNA damage response processes, positioning DNA damage as an important avenue for further research of TEAD proteins.

Transcriptional integration of mitogenic and mechanical signals by Myc and YAP.

Mammalian cells must integrate environmental cues to determine coherent physiological responses. The transcription factors Myc and YAP-TEAD act downstream from mitogenic signals, with the latter responding also to mechanical cues. Here, we show that these factors coordinately regulate genes required for cell proliferation. Activation of Myc led to extensive association with its genomic targets, most of which were prebound by TEAD. At these loci, recruitment of YAP was Myc-dependent and led to full transcriptional activation. This cooperation was critical for cell cycle entry, organ growth, and tumorigenesis. Thus, Myc and YAP-TEAD integrate mitogenic and mechanical cues at the transcriptional level to provide multifactorial control of cell proliferation.

Mutual dependence of the MRTF-SRF and YAP-TEAD pathways in cancer-associated fibroblasts is indirect and mediated by cytoskeletal dynamics.

Both the MRTF-SRF and the YAP-TEAD transcriptional regulatory networks respond to extracellular signals and mechanical stimuli. We show that the MRTF-SRF pathway is activated in cancer-associated fibroblasts (CAFs). The MRTFs are required in addition to the YAP pathway for CAF contractile and proinvasive properties. We compared MRTF-SRF and YAP-TEAD target gene sets and identified genes directly regulated by one pathway, the other, or both. Nevertheless, the two pathways exhibit mutual dependence. In CAFs, expression of direct MRTF-SRF genomic targets is also dependent on YAP-TEAD activity, and, conversely, YAP-TEAD target gene expression is also dependent on MRTF-SRF signaling. In normal fibroblasts, expression of activated MRTF derivatives activates YAP, while activated YAP derivatives activate MRTF. Cross-talk between the pathways requires recruitment of MRTF and YAP to DNA via their respective DNA-binding partners (SRF and TEAD) and is therefore indirect, arising as a consequence of activation of their target genes. In both CAFs and normal fibroblasts, we found that YAP-TEAD activity is sensitive to MRTF-SRF-induced contractility, while MRTF-SRF signaling responds to YAP-TEAD-dependent TGFß signaling. Thus, the MRF-SRF and YAP-TEAD pathways interact indirectly through their ability to control cytoskeletal dynamics.

Substrate stiffness promotes vascular smooth muscle cell calcification by reducing the levels of nuclear actin monomers.

BACKGROUND: Vascular calcification (VC) is a prevalent independent risk factor for adverse cardiovascular events and is associated with diabetes, hypertension, chronic kidney disease, and atherosclerosis. However, the mechanisms regulating the osteogenic differentiation of vascular smooth muscle cells (VSMC) are not fully understood. METHODS: Using hydrogels of tuneable stiffness and lysyl oxidase-mediated stiffening of human saphenous vein ex vivo, we investigated the role of substrate stiffness in the regulation of VSMC calcification. RESULTS: We demonstrate that increased substrate stiffness enhances VSMC osteogenic differentiation and VSMC calcification. We show that the effects of substrate stiffness are mediated via a reduction in the level of actin monomer within the nucleus. We show that in cells interacting with soft substrate, elevated levels of nuclear actin monomer repress osteogenic differentiation and calcification by repressing YAP-mediated activation of both TEA Domain transcription factor (TEAD) and RUNX Family Transcription factor 2 (RUNX2). CONCLUSION: This work highlights for the first time the role of nuclear actin in mediating substrate stiffness-dependent VSMC calcification and the dual role of YAP-TEAD and YAP-RUNX2 transcriptional complexes.

YAP/TAZ-TEAD signalling axis: A new therapeutic target in malignant pleural mesothelioma.

The Hippo signalling pathway, a highly conserved signalling cassette, regulates organ size by controlling cell growth, apoptosis and stem cell self-renewal. The tumourigenic potential of this pathway is largely attributed to the activity of YAP/TAZ, which activate the TEAD1-4 transcription factors, leading to the expression of genes involved in cell proliferation and suppression of cell death. Aberrant regulation of the YAP/TAZ-TEAD signalling axis is commonly observed in malignant pleural mesothelioma (MPM), an insidious neoplasm of the pleural tissue that lines the chest cavity and covers the lungs with poor prognosis. Given the limited effectiveness of current treatments, targeting the YAP/TAZ-TEAD signalling cascade has emerged as a promising therapeutic strategy in MPM. Several inhibitors of the YAP/TAZ-TEAD signalling axis are presently undergoing clinical development, with the goal of advancing them to clinical use in the near future.

The YAP-TEAD4 complex promotes tumor lymphangiogenesis by transcriptionally upregulating CCBE1 in colorectal cancer.

The secreted protein collagen and calcium-binding EGF domain 1 (CCBE1) is critical for embryonic lymphatic development through its role in the proteolytic activation of mature vascular endothelial growth factor C (VEGFC). We previously reported that CCBE1 is overexpressed in colorectal cancer (CRC) and that its transcription is negatively regulated by the TGFß-SMAD pathway, but the transcriptional activation mechanism of CCBE1 in CRC remains unknown. Recent studies have revealed the vital role of the hippo effectors YAP/TAZ in lymphatic development; however, the role of YAP/TAZ in tumor lymphangiogenesis has not been clarified. In this study, we found that high nuclear expression of transcription factor TEAD4 is associated with lymph node metastasis and high lymphatic vessel density in patients with CRC. YAP/TAZ-TEAD4 complexes transcriptionally upregulated the expression of CCBE1 by directly binding to the enhancer region of CCBE1 in both CRC cells and cancer-associated fibroblasts, which resulted in enhanced VEGFC proteolysis and induced tube formation and migration of human lymphatic endothelial cells in vitro and lymphangiogenesis in a CRC cell-derived xenograft model in vivo. In addition, the bromodomain and extraterminal domain (BET) inhibitor JQ1 significantly inhibited the transcription of CCBE1, suppressed VEGFC proteolysis, and inhibited tumor lymphangiogenesis in vitro and in vivo. Collectively, our study reveals a new positive transcriptional regulatory mechanism of CCBE1 via YAP/TAZ-TEAD4-BRD4 complexes in CRC, which exposes the protumor lymphangiogenic role of YAP/TAZ and the potential inhibitory effect of BET inhibitors on tumor lymphangiogenesis.

Inhibition of transcriptional coactivator YAP Impairs the expression and function of transcription factor WT1 in diabetic podocyte injury.

Podocyte injury and loss are hallmarks of diabetic nephropathy (DN). However, the molecular mechanisms underlying these phenomena remain poorly understood. YAP (Yes-associated protein) is an important transcriptional coactivator that binds with various other transcription factors, including the TEAD family members (nuclear effectors of the Hippo pathway), that regulate cell proliferation, differentiation, and apoptosis. The present study found an increase in YAP phosphorylation at S127 of YAP and a reduction of nuclear YAP localization in podocytes of diabetic mouse and human kidneys, suggesting dysregulation of YAP may play a role in diabetic podocyte injury. Tamoxifen-inducible podocyte-specific Yap gene knockout mice (YappodKO) exhibited accelerated and worsened diabetic kidney injury. YAP inactivation decreased transcription factor WT1 expression with subsequent reduction of Tead1 and other well-known targets of WT1 in diabetic podocytes. Thus, our study not only sheds light on the pathophysiological roles of the Hippo pathway in diabetic podocyte injury but may also lead to the development of new therapeutic strategies to prevent and/or treat DN by targeting the Hippo signaling pathway.