The circadian clock protein Cryptochrome 1 is a direct target and feedback regulator of the Hippo pathway.

Circadian clock controls daily behavior and physiology. The activity of various signaling pathways affects clock gene expression. Here, we show that the core circadian clock gene CRY1 is a direct target of the Hippo pathway effector YAP. YAP binds to TEADs and occupies the proximal promoter regions of CRY1, positively regulating its transcription. Interestingly, we further identified that CRY1 acts in a feedback loop to fine-tune Hippo pathway activation by modulating the expression of YAP and MOB1. Indeed, loss of CRY1 results in enhanced YAP activation. Consistently, we found that YAP levels and activity control clock gene expression and oscillation in synchronized cells. Furthermore, in breast cancer cells, CRY1 downregulation causes YAP/TAZ hyperactivation and enhanced DNA damage. Together, our findings provide a direct mechanistic link between the Hippo pathway and the circadian clock, where CRY1 and Hippo components form an orchestrated signaling network that influences cell growth and circadian rhythm.

Pharmacological blockade of TEAD-YAP reveals its therapeutic limitation in cancer cells.

Targeting TEAD autopalmitoylation has been proposed as a therapeutic approach for YAP-dependent cancers. Here we show that TEAD palmitoylation inhibitor MGH-CP1 and analogues block cancer cell "stemness", organ overgrowth and tumor initiation in vitro and in vivo. MGH-CP1 sensitivity correlates significantly with YAP-dependency in a large panel of cancer cell lines. However, TEAD inhibition or YAP/TAZ knockdown leads to transient inhibition of cell cycle progression without inducing cell death, undermining their potential therapeutic utilities. We further reveal that TEAD inhibition or YAP/TAZ silencing leads to VGLL3-mediated transcriptional activation of SOX4/PI3K/AKT signaling axis, which contributes to cancer cell survival and confers therapeutic resistance to TEAD inhibitors. Consistently, combination of TEAD and AKT inhibitors exhibits strong synergy in inducing cancer cell death. Our work characterizes the therapeutic opportunities and limitations of TEAD palmitoylation inhibitors in cancers, and uncovers an intrinsic molecular mechanism, which confers potential therapeutic resistance.

Discovery of a new class of reversible TEA domain transcription factor inhibitors with a novel binding mode.

The TEA domain (TEAD) transcription factor forms a transcription co-activation complex with the key downstream effector of the Hippo pathway, YAP/TAZ. TEAD-YAP controls the expression of Hippo-responsive genes involved in cell proliferation, development, and tumorigenesis. Hyperactivation of TEAD-YAP activities is observed in many human cancers and is associated with cancer cell proliferation, survival, and immune evasion. Therefore, targeting the TEAD-YAP complex has emerged as an attractive therapeutic approach. We previously reported that the mammalian TEAD transcription factors (TEAD1-4) possess auto-palmitoylation activities and contain an evolutionarily conserved palmitate-binding pocket (PBP), which allows small-molecule modulation. Since then, several reversible and irreversible inhibitors have been reported by binding to PBP. Here, we report a new class of TEAD inhibitors with a novel binding mode. Representative analog TM2 shows potent inhibition of TEAD auto-palmitoylation both in vitro and in cells. Surprisingly, the co-crystal structure of the human TEAD2 YAP-binding domain (YBD) in complex with TM2 reveals that TM2 adopts an unexpected binding mode by occupying not only the hydrophobic PBP, but also a new side binding pocket formed by hydrophilic residues. RNA-seq analysis shows that TM2 potently and specifically suppresses TEAD-YAP transcriptional activities. Consistently, TM2 exhibits strong antiproliferation effects as a single agent or in combination with a MEK inhibitor in YAP-dependent cancer cells. These findings establish TM2 as a promising small-molecule inhibitor against TEAD-YAP activities and provide new insights for designing novel TEAD inhibitors with enhanced selectivity and potency.

Regulation of Cell Polarity by Posttranslational Protein Palmitoylation.

Cell polarity is a common feature of many living cells, especially epithelial cells, and plays important roles in development, tissue homeostasis, and diseases. Therefore, the signaling pathways involved in establishing and maintaining cell polarity are tightly controlled. Protein S-palmitoylation has been recently recognized as an important posttranslational modification involved in cell polarity, via dynamic covalent attachment of fatty acyl groups to the cysteine residues of cell polarity proteins. Here, we describe the methods to study the function and regulation of S-palmitoylation of cell polarity proteins.