Hippo Pathway in Cancer: Aberrant Regulation and Therapeutic Opportunities

Hippo Pathway in Cancer: Aberrant Regulation and Therapeutic Opportunities

Trends in Cancer Review Hippo Pathway in Cancer: Aberrant Regulation and Therapeutic Opportunities Philamer C. Calses,1,2 James J. Crawford,3 Jennie...

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Trends in Cancer

Review

Hippo Pathway in Cancer: Aberrant Regulation and Therapeutic Opportunities Philamer C. Calses,1,2 James J. Crawford,3 Jennie R. Lill,2 and Anwesha Dey1,* The Hippo pathway remains a central focus in both basic and translational research and is a key modulator of developmental biology. Dysregulation of the pathway is associated with a plethora of human cancers and there are multiple efforts to target key components of the pathway for disease intervention. In this review, we briefly highlight the latest research advances around the core components of the Hippo pathway in cancer. More specifically, we discuss several genetic aberrations of these factors as mechanisms for the development of cancers, including genetic amplification, deletion, and gene fusions. Additionally, we highlight the role of the Hippo pathway in cancer therapy resistance and tumor immunogenicity. Last, we summarize the ongoing efforts to target the pathway in cancers.

Highlights The Hippo pathway is a well-conserved signaling pathway across higher-order vertebrates that regulates organ size and tissue homeostasis. The Hippo pathway can be deregulated leading to oncogenesis and chemotherapeutic resistance through a variety of mechanisms including amplification, mutations in upstream signaling factors, and gene fusions. Multiple efforts to target key regulators of the Hippo pathway for intervention in tumorigenesis have been reported and these are associated with the targeting of YAP/TAZ and TEAD interactions, which are inherently difficult to target.

The Hippo Pathway The Hippo pathway is a highly conserved signaling pathway across higher-order vertebrates that modulates key target genes to regulate a multitude of biological processes including cellular proliferation, survival, differentiation, cellular fate determination, organ size, and tissue homeostasis (Figure 1). At the core of the pathway are serine/threonine kinases, sterile 20-like kinase 1/2 (MST1/2), and large tumor suppressor 1/2 (LATS1/2). Recently, MAP4K and TAOK kinases have been shown to directly phosphorylate LATS1/2, thus acting in parallel with MST1/2 [1]. These kinases, along with the adaptor proteins, Salvador homolog 1 (SAV1) and MOB kinase activator 1A/B (MOB1A/B), phosphorylate and inhibit downstream effector proteins, Yesassociated protein (YAP1), and its paralog transcriptional coactivator with PDZ-binding motif (TAZ) (also known as WWTR1) and sequestrates them in the cytoplasm by binding to 14-3-3 proteins [2]. Notably, the tumor suppressor neurofibromin 2 (NF2) (also known as Merlin) participates upstream of these kinases to inhibit YAP and TAZ activity by promoting the activation of the pathway. Additional phosphorylation of YAP/TAZ leads to proteasome-mediated degradation facilitated by binding to β-TrCP [3,4]. Such regulation prevents YAP/TAZ from accumulating in the nucleus and from binding to a family of sequence-specific transcription factors called TEA DNA-binding proteins (TEAD1–4) that mediate proliferative and prosurvival genes such as CTGF, CRY61, BIRC5, ANKRD1, and AXL (Figure 2). Besides TEADs, YAP/TAZ also cooperates with RUNT-related transcription factors (RUNX1 and 2), T-box transcription factor 5 (TBX5), and SMADs as among others [2,5].

Hippo Pathway Deregulation in Cancer Overexpression of Hippo Pathway Effector Proteins Aberration of the Hippo pathway is associated with the hallmarks of oncogenesis and, more recently, has been linked to other cellular processes such as regulation of T cell functionality [6]. These hallmarks include the induction of hyperproliferation, cellular invasion, and metastasis, as well as a role in cancer cell maintenance and chemotherapeutic resistance mechanisms. Analysis of over 9000 tumors showed that YAP and TAZ are frequently amplified in head and neck and gynecologic cancers, while the upstream regulators LATS1/2 and NF2 are commonly mutated

1

Department of Discovery Oncology, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA 2 Department of Microchemistry, Proteomics, and Lipidomics, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA 3 Department of Discovery Chemistry, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA

*Correspondence: [email protected] (A. Dey).

Trends in Cancer, May 2019, Vol. 5, No. 5 https://doi.org/10.1016/j.trecan.2019.04.001 © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Figure 1. The Hippo Pathway. The Hippo pathway was initially established in Drosophila melanogaster as a key modulator of organ size. This highly conserved signaling pathway regulates many biological processes including cellular and immune homeostasis, regeneration and repair, and cancer development.

in gastrointestinal and gynecologic cancers and mesotheliomas [7,8]. Similarly, Wang et al. recently reported a comprehensive analysis of The Cancer Genome Atlas of 19 Hippo core genes, which revealed that, from 33 cancer types, YAP and TAZ had the highest amplification frequency in squamous cancers. Among these, YAP and TAZ were mutually amplified in head and neck squamous cell carcinoma (HNSC) and cervical squamous cell carcinoma (CESC) [8]. Moreover, gain/loss-of-function mutations in YAP and TAZ, although very rare, were shown to be functionally important [8]. As expected, mutations of the upstream factors of the Hippo pathway are mutually exclusive with YAP and TAZ amplification [8]. These studies and others discussed elsewhere provide a framework for which cancer indications should be prioritized for targeting the Hippo pathway in cancers. In line with the observation that mutations are highly associated with various malignancies, additional studies show that overexpression of nuclear YAP/TAZ and elevated TEAD expression correlate with poor prognostic outcome and increased therapeutic resistance in most cancer types that arise from thoracic, gastric, genitourinary, gynecologic, skin, bone, or brain [9]. While some of these prognostic outcomes are because of YAP/TAZ/TEAD amplifications, the mechanism of overexpression remains largely unknown. Recent progress suggests the cooperation 298

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Figure 2. The Core of the Hippo Pathway and Opportunities for Drugging the Pathway in Cancer. (A) The Hippo pathway transcriptionally regulates target genes involved in cell fate determination, polarity, proliferation, and survival. Neurofibromatosis 2 (NF2) induces the activation of serine/threonine kinases, large tumor suppressor 1/2 (LATS1/2) and sterile 20-like kinase ½ (MST1/2), that in turn phosphorylate Yes-associated protein (YAP) and transcriptional coactivator with PDZbinding motif (TAZ). YAP and TAZ are subsequently sequestered in the cytoplasm and degraded by the proteasome. Recently, studies have shown that the Hippo pathway plays a role in cancer immunotherapy. (B) Approaches for drugging the Hippo pathway in cancer. These include approaches to inhibit the nuclear translocation of YAP and TAZ, promote degradation of YAP and TAZ and inhibit the interaction between YAP/TAZ and TEAD1–4.

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of additional regulators, including crosstalk with chromatin remodelers and other signaling pathways. A recent study demonstrated that coamplification of the chromatin remodeler ACTL6A, and p63, transcriptionally increases YAP expression thereby increasing cellular proliferation, and that this amplification event strongly correlates with poor prognosis in patients with head and neck squamous cell carcinoma (HNSCC) [10]. Recently, YAP was also shown to undergo monomethylation by SET1A, which in turn blocks its cytoplasmic export and ultimately promotes tumorigenesis [11]. As part of a crosstalk with other signaling pathways, YAP nuclear localization and activation is induced by PI3 kinase (PI3K) and phosphoinositide-dependent kinase (PDK1), which work in concert by inhibiting the Hippo pathway, leading to poor prognosis in patients [12,13]. While discussed elsewhere, it is important to note that other signaling pathways, including WNT and G protein-coupled signaling, as well as processes like metabolism and mechanotransduction also regulate YAP/TAZ [14]. Disruption of Upstream Regulators Disruption, and more specifically loss of the upstream tumor suppressors can lead to YAP nuclear activation, which in turn promotes tumorigenesis. Consistent with this, YAP nuclear localization is strongly associated with human NF2-mutant tumors arising from the nervous system such as schwannomas, meningiomas, and ependymomas [9]. In mice, Nf2 loss cooperates with Ras to induce Yap activation as a novel mechanism to induce thyroid cancers [15]. Interestingly, activating mutations of GNAQ and GNA11, which act upstream of the Hippo pathway, are exclusively found in 60% of uveal melanoma and activate YAP-dependent cell growth independent of the Hippo pathway [16,17]. Recently, genetic alterations and methylation of the tumor suppressor LATS1/2 has been observed in several cancers [7]. However, it is largely unknown whether this observation is dependent on YAP growth-promoting activity. Interestingly, with the exception of NF2, hotspots or point mutations are rarely observed in the core components of the Hippo pathway in cancers. Gene Fusions Another mutation found in the Hippo pathway is gene fusions. These are structural chromosomal aberrations that often involve the exchange of regulatory regions of genes and are strong driver mutations involved in tumorigenesis. Core factors of the Hippo pathway have been found to contain such anomalies. Notably, the TAZ-CAMTA1 (TC) gene fusion is the initiating mutation that accounts for greater than 90% of a vascular cancer called epithelioid hemangioendothelioma (EHE) [18]. TC functions similarly to wild-type TAZ in that it transcriptionally activates TEAD-dependent target genes. However, unlike wild-type TAZ, whose nucleocytoplasmic transport is regulated by the Hippo pathway, TC is constitutively expressed in the nucleus [18]. Interestingly, another gene fusion was observed with YAP and the transcription factor E3 (TFE3) [19]. Additionally, gene fusions were found in TAZ, NF2, and LATS1/2 in lung cancer [20]. It is intriguing to speculate whether these genetic features are due to the inherent genomic instability associated with cancer or are driven by some other mechanisms. It is also unknown whether these specific fusions are found broadly in cancer and whether they have the capability to function independently of the Hippo pathway or whether they function through TEADs. Furthermore, it is interesting to determine whether these gene fusions would complicate the ongoing efforts to drug the pathway and whether these gene fusions function as a resistance mechanism against cancer therapies. Intrinsic and Acquired Resistance Chemotherapy remains the most standard course of treatment for cancer patients. However, a significant number of tumors are resistant to chemotherapeutic treatment by two mechanisms: intrinsic (preexisting) or acquired resistance, both of which can lead to tumor relapse and patient mortality. Deregulation of signaling pathways has been implicated as a mechanism 300

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of chemotherapy resistance, including the Hippo pathway (covered in detail by Nguyen and Li in this issue). For instance, increased YAP/TAZ expression and activity has been suggested to promote Taxol resistance in several cancer cell lines perhaps by maintaining a population of cancer stem cells, at least in the case of breast cancer [21,22]. While differential YAP phosphorylation by different kinases has been extensively characterized [1,5], this model proposes that YAP/TAZ is phosphorylated by cyclin-dependent kinase 1 (CDK1) independent of the Hippo pathway and leads to increased apoptosis and cellular sensitivity to Taxol treatment [23,24]. It would be interesting to determine whether CDK1 is inactive in YAP/TAZdependent Taxol resistance, because it is been shown that at least YAP was not phosphorylated in Taxol-resistant cancer cell lines [24]. Additionally, YAP/TAZ has been implicated in other forms of chemotherapeutic resistance, particularly in the case of DNA-damaging agents such as doxorubicin and 5-fluorouracil (5-FU) [25,26]. Although the mechanism by which LATS1/2 mediates chemotherapeutic resistance is unknown, it is worth noting that depletion of LATS1/2 was identified from a functional genomics screen to cause Taxol resistance [27]. Additionally, LATS2 expression is markedly decreased in highly resistant leukemic cells [28]. Similarly, MST1 protein expression was decreased through degradation in cisplatin-resistant prostate cancer cells [29]. Taken together, these studies reveal that components of the Hippo pathway mediate a path to chemotherapeutic resistance. However, further studies are warranted to determine why and how the Hippo pathway is involved and whether this is context dependent. While chemotherapy for malignant intervention is still often used, pathway-targeted therapies are a rapidly expanding therapeutic approach to selectively treat patients with various types of cancer associated with mutations in key oncogenic drivers. It has become clear that intrinsic and acquired resistance to these targeted therapies is also a growing concern. For example, several mechanisms of resistance have been proposed for therapies against epidermal growth factor receptor (EGFR) and its downstream effectors [30]. Activating mutations of EGFR, RAS, and BRAF are frequently observed and are the most common targets for targeted therapies [31]. The Hippo pathway has recently been implicated in mediating resistance to various targeted therapies. YAP activation, for example, has been cited as the culprit in bypassing pathway-targeted therapies. For instance, in EGFR-mutant lung cancer cells, YAP activation was identified to induce the AXL tyrosine kinase receptor as a mechanism of intrinsic and acquired resistance, although the prevalence in patients is largely unknown [32,33]. Notably, in a mouse model harboring an inducible oncogenic Kras-G12D mutation that drives pancreatic ductal adenocarcinoma (PDAC), knockdown of Kras led to complete tumor regression. Eventually, a significant number of these mice spontaneously relapsed despite apparent ablation of Kras due to Yap amplification and activation, thereby bypassing oncogenic Kras withdrawal [34]. Consistent with this study, YAP was identified from a gain-of-function screen as a key driver that replaces RAS signaling in KRASdependent colon cancer cell lines [35]. While both studies revealed an important role of YAP in bypassing oncogenic KRAS, downstream transcriptional outputs differ in that TEAD2 mediates growth in one study and in the other study this is mediated by FOS. Thus, depending on the cellular context, different transcriptional programs are activated. Nevertheless, these studies indicate that the mechanisms of therapy resistance in KRASdriven tumors are context dependent, but places YAP at the center of, or at least as a part of, various mechanisms. YAP activation has also been shown to confer resistance to RAF and MEK therapies [36]. Consistently, YAP activation was found to bypass therapy by promoting survival by upregulation of the antiapoptotic gene BCL-xL in BRAF-mutant cells [36]. YAP and TAZ were among the

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top candidates from a large-scale functional genetic study that aimed to identify mechanisms of resistance to ALK inhibition in ALK-addicted lung cancer [37]. In most of these cases, a combinatorial regimen with YAP inhibition along with targeted therapy of these other pathways overcomes intrinsic and acquired therapeutic resistance. Thus, elucidation of the mechanism of YAP activation during drug therapy resistance is paramount when considering targeted therapies in cancer. These studies raise the intriguing possibility of whether the Hippo pathway plays a broader or more specific role in mediating cancer therapy resistance across various cancer types. This topic warrants further investigation in addition to elucidation of the underlying mechanisms. The Role of the Hippo Pathway and Tumor Immunogenicity Recent studies have uncovered an interesting role associating the Hippo pathway with tumor immunity. Using syngeneic mouse tumor models, Moroishi et al. found that Lats1/2 loss inhibited tumor growth. Mechanically, Lats1/2-depleted tumor cells secreted nucleic acidrich extracellular vesicles that induce an interferon type 1 response to activate T cells, thereby improving tumor immunogenicity and tumor destruction [38]. Interestingly, MST1/2 was found to be mutated in a rare form of human combined immunodeficiency (CID) that resulted in increased apoptosis of naïve and proliferating T cells [39]. However, future studies need to be performed to elucidate the implications of these findings in human cancers. In contrast to these earlier studies, Ni et al. demonstrated that YAP is highly expressed and functions in the generation of regulatory T cells (Tregs), which are implicated in the suppression of antitumor immunity and are enriched in many tumors [6]. More specifically, YAP induces the expression of Activin by upregulating TGFβ/SMAD signaling. Interestingly, the Hippo pathway has also been shown to modulate the immune checkpoint molecule PD-L1 [40]. Depletion of MST1/2 or LATS1/2, in conjunction with overexpression of constitutively active YAP or TAZ, increases PD-L1 expression in breast and lung cancer cell lines [40]. Here, TAZ exerts a prominent role in increasing PD-L1 expression in cancer cells, thus promoting immune cell evasion, although it is worth highlighting that this was reported to be species dependent [40]. Similarly, PD-L1 expression is induced by YAP in BRAF-inhibitor-resistant melanoma, which mediates the evasion from cytotoxic T cells [41]. Another study demonstrated that TAZ plays an important role in regulating the differentiation of T helper and Treg cells [42]. Given these various findings, alternative therapies targeting the Hippo pathway in cancer cells or immune cells may pose some dichotomous challenges. Before any clinical or translational relevance is determined, further studies need to be performed to elucidate whether the role of the core Hippo pathway members in different immune cells is context dependent. This is undoubtedly an area of active investigation in the field.

Targeting the Hippo Pathway in Cancer Given its broad potential therapeutic relevance, it is unsurprising that there is significant interest in targeting the Hippo pathway, with cancer seemingly front and center in terms of disease indications of interest. When considering how one might go about this, some potential issues immediately come to mind. Upstream of the key pathway effectors is the core kinase cascade MST1/2 and LATS1/2, which, along with NF2, are bona fide tumor suppressors [43]. Inhibition of any of these, perhaps with the exception of LATS1/2 in the context of immunotherapy, might serve to increase transcriptional activity and thus be counterproductive. Efforts to identify inhibitors of MST1/2 have been reported but have yet to yield drug-like molecules for clinical evaluation [38,44]. In addition, a recent patent application reported the first targeted inhibitors of LATS1/2 [45]. Taking these aside, this leaves YAP and TAZ, the transcriptional coactivators of the pathway along with the TEADs at the forefront in targeting the Hippo pathway.

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There are key potentially druggable sites in the protein–protein interaction between YAP/TAZ and TEAD that have been identified, as well as a highly conserved palmitoylation pocket in TEADs [46–49]. These provide a platform to screen for small molecules to target the Hippo pathway in cancers. One such small molecule is verteporfin (Table 1, compound 1) also known as Visudyne. Along with other, structurally related porphorins, verteporfin has been widely reported as a YAP inhibitor, and features prominently in the Hippo pathway literature [50]. Verteporfin, an FDA-approved drug for the treatment of wet aged-macular degeneration (AMD), is a porphyrin derivative that acts as a photosensitizer, causing its primary biological activity via the action of short-lived singlet oxygen and reactive oxygen radicals. While verteporfin treatment has been reported to reduce the expression of mRNAs of known Hippo pathway target genes such as CYR61, CTGF, AXL, and BIRC-5, it is worth noting that it is also known to induce oligomerization of proteins implicated in major cellular processes, including autophagy and cytoskeletal maintenance among others [51,52]. Thus, its suitability as a starting point for drug discovery efforts, as well as its usefulness as a tool compound, is unclear. In a similar vein, CA3 (Table 1, compound 2), was reported as a YAP inhibitor that can modulate YAP/TEAD transcriptional activity and decrease YAP expression [53]. However, like Verteporfin, its mode of inhibition and mechanism of interaction with YAP remain unknown. Structurally similar fluorene–oxime compounds (Table 1, compound 3) have been proposed as Hippo pathway inhibitors by Vivace Therapeutics based on activity in a YAP reporter assay [54,55]. Taking the concept further downstream to the node of the pathway, a recent review summarized published patent applications that claim direct small-molecule inhibitors of the YAP/TAZ–TEAD interaction [56]. A French biotechnology company, Inventiva, has filed multiple patent applications based on a bis-aryl hydrazine scaffold (Table 1, compound 4), with modest activity in a TEAD– GAL4 transactivation assay and antiproliferative activity in the NCI-H2052 mesothelioma cell line, albeit at high concentrations [57,58]. The other report of YAP/TEAD inhibitors focuses on a more defined, if less direct, method of inhibition that targets a lipid pocket at the core of all four TEADs, which is generally occupied by a palmitoyl ligand and is essential for TEAD folding, stability, and YAP binding [46–48]. The representative example that seems to have been most extensively characterized is compound 5 (Table 1, compound 5), which was shown to inhibit target gene expression and cell proliferation in a liver cell line, HuH7 [59]. However, activity was reported only at relatively high concentrations (30 and 10 μM, respectively). Interestingly, flufenamic acid (Table 1, compound 6), a nonsteroidal anti-inflammatory drug (NSAID), binds to the central pocket of TEAD2 but did not inhibit YAP– TEAD binding [60]. Flufenamic acid decreased cell growth, TEAD reporter activity, and several Hippo pathway responsive genes. However, the underlying mechanism remains unclear. More recently, an independent group identified flufenamic acid derivatives (Table 1, compound 7) comprising chloromethyl ketone moieties that bind to the conserved cysteine in the lipid pocket, thus inhibiting YAP–TEAD interaction, transcriptional activity, and glioblastoma growth in vitro [61]. In an early report in this emerging field, Hu and coworkers at Roche identified cyclic YAP-like peptide YAP–TEAD blockers [62]. Using structure-based design and employing a disulfide bridge as a surrogate for a cation − π interaction and peptide conformational constraint they identified (Table 1, compound 8) an inhibitor with an IC50 of 25 nM. This peptide was able to compete with endogenous YAP binding to GST-TEAD1 in cell lysate and thus represents a useful tool molecule. However, in that study the researchers were unable to leverage this into a cell-active tool compound. Jiao et al. found an approach to targeting YAP–TEAD by developing a peptide mimicking VGLL4, known as ‘super-TDU’ [63] (Table 1, compound 9). This comes from the understanding that, like YAP and TAZ, a family of proteins called Vestigial-like protein (VGLL) 1–4 bind to TEADs [64]. While VGLL1–3 have been shown to act as transcription coactivators, like Trends in Cancer, May 2019, Vol. 5, No. 5

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Table 1. Structures of Representative Compounds Reported to Modulate Hippo Pathway Activity with Their Source and Proposed Mechanism of Action Compound

Reported Mechanism of Action

Institution

Refs

-YAP–TEAD inhibitor -Mechanism is unclear as multiple targets reported

Bausch +Lomb Visudyne is a trademark of Novartis AG

[50–52]

-Decreases YAP expression -Mechanism is unclear

MD Anderson Cancer Center

[53]

-YAP/TAZ–TEAD inhibitor -Mechanism is unclear from patent

Vivace Therapeutics

[54–55]

-YAP/TAZ–TEAD inhibitor -Mechanism is unclear from patent

Inventiva

[57–58]

-Targets palmitoylation pocket of TEADs -Mechanism is unclear from patent

Mass General Hospital

[59]

-Targets palmitoylation pocket of TEADs -TEAD2–YAP binding unaffected

A*STAR

[60]

-Targets palmitoylation pocket of TEADs -TEAD4–YAP binding is reduced

Indiana Universitys

[61]

-YAP–TEAD binding competitor -TEAD1–YAP binding is inhibited

Roche

[62]

-YAP–TEAD binding competitor -VGLL4 peptide binds to TEADs -TEAD4–YAP binding is reduced

Chinese Academy of Sciences

[63]

1.

2.

3.

4.

5.

6.

7.

8.

9. SVDDHFAKSLGDTWLQIGGSGNPKTANVPQTVPMRLRKLPDSFFKPPE

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YAP and TAZ, VGLL4 behaves as a tumor suppressor and is downregulated in gastric cancers [63]. Using this peptide, growth of gastric cells in vitro and in vivo in mice was significantly reduced. Mechanistically, ‘super-TDU’ peptides bind to TEAD and compete with YAP binding [63]. While this provides proof of concept for these types of antagonistic peptides, it remains to be determined whether these are clinically viable and whether they are broadly applicable in other cancers. Other compounds reported to target the Hippo pathway by various direct or indirect measures have recently been reviewed elsewhere and represent an area of active investigation [65]. Although none of the currently reported inhibitors have advanced to the clinical stage yet, to the best of our knowledge, they offer promising proof of concept in terms of targeting the Hippo pathway across multiple disease areas.

Concluding Remarks The Hippo pathway, particularly YAP/TAZ and TEADs, are key players in cancer development. While the biology continues to unravel, there is sufficient evidence that this is a key pathway worth targeting in oncology, and there are multiple efforts around the world working towards this goal. Looking ahead, other approaches to target the YAP/TAZ–TEAD complex can be exploited by the relevant biology, such as identifying negative regulators of YAP and TAZ. It is well known that YAP and TAZ contain phosphodegron sites, which on activation by the Hippo pathway and other kinases lead to proteasomal degradation [3,4]. In a less studied context, YAP protein is also degraded through the lysosome as part of the innate antiviral immunity response; however, further studies are warranted on whether this biology is relevant in cancers [66]. As a proof of concept, perhaps one can take a high-throughput approach in screening through small molecules that specifically induce the phosphodegron on YAP and TAZ. Additionally, since YAP and TAZ undergo nucleocytoplasmic transport, the identification of small molecules that inhibit their nuclear transport would provide another approach in negatively regulating YAP and TAZ. For example, dasatinib, statins, and pazopanib inhibited the nuclear translocation of YAP and TAZ in breast cancer cell lines [67]. In this study, Oku et al. further showed that treatment with these compounds induced the proteasomal degradation of YAP and TAZ as well as decreased cellular proliferation in combination of these compounds or with chemotherapy. While some of the regulation of these factors at the protein level is well characterized and mediated mostly by phosphorylation, other post-translational modifications that negatively regulate YAP, TAZ, and TEADs remain unexplored.

Outstanding Questions What regulates the transcription of YAP/ TAZ–TEADs in cancer and under normal physiological conditions? Which transcription factors synergistically cooperate with YAP/TAZ–TEADs to promote cancer and is this synergy tissue or context specific? What determines which factors cooperate with YAP/TAZ–TEADs in various contexts? Are there additional context-specific extracellular signals to activate YAP/ TAZ–TEADs in cancer? Are there additional receptors and ligands that directly regulate and are dedicated to the Hippo pathway? What are the negative regulators of YAP/ TAZ–TEADs? Could targeting of these factors be exploited as a potential agonist of the pathway? What is the best mechanism for targeting YAP/TAZ–TEADs in cancer?

Another avenue that is less defined in targeting YAP, TAZ, and TEADs in cancer is to elucidate what regulates these genes at the transcriptional and post-transcriptional levels. While it is currently unknown what regulates these genes transcriptionally, several studies have shown that several small noncoding RNAs called miRNAs negatively regulate YAP, TAZ, and TEAD3 [8,68,69]. Moreover, nuclear YAP has been shown to sequester p72, a regulatory component of the miRNA-processing machinery, and suppresses global miRNA expression in skin and liver tumor models in mice [70], thus highlighting the need to inhibit YAP in cancers. Finally, crosstalk of the Hippo pathway with other signaling pathways and molecules could offer additional avenues to target this pathway in cancers [5]. While this review has focused mainly on opportunities for directly targeting the core Hippo pathway members/transcription factors, eventually combination therapies with molecules that could directly/indirectly modulate the pathway, like inhibitors of PI3K, GPCRs, and EGFR to name a few, could be promising strategies. The fact remains that a well-characterized in vitro and in vivo tool compound remains elusive, at least for now. Drugging of protein–protein interactions is inherently difficult to accomplish due to Trends in Cancer, May 2019, Vol. 5, No. 5

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the additional difficulties in finding and optimizing ligands that are both interaction disruptive and specific. Moreover, targeting transcription factors adds an additional layer of complexity, especially those involved in cellular homeostasis. As a consequence, one of the key challenges ahead in the therapeutic targeting of a pathway as broadly active as the Hippo pathway is safety, more so than efficacy. Given that there are four TEADs (1–4), a potent inhibitor targeting the pathway might require a pan-TEAD inhibitor that blocks the interaction of all four TEADs with YAP and TAZ, and this is likely to be no small feat. Thus, unless the relevant biology of each can be parsed out, and sufficiently paralog-selective ligands developed, this will require significant research efforts. Additional questions (outlined below and see Outstanding Questions) will need to be answered specifically for each disease area. These might include: (i) what are the relevant biomarkers; (ii) what are the key indications, such as in oncology, that would benefit most from targeting the pathway; (iii) what determines genetic dependency on the pathway in cancers; and (iv) what targeting strategy will prove to be the safest and most efficacious? One thing that seems clear is that given the recent rapid advances in all aspects, the future is undoubtedly promising for drugging the Hippo pathway in cancers and beyond. References 1.

2.

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4.

5. 6.

7. 8.

9. 10.

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12.

13.

14. 15.

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