Alternative Wnt Signaling Activates YAP/TAZ.

The transcriptional co-activators YAP and TAZ are key regulators of organ size and tissue homeostasis, and their dysregulation contributes to human cancer. Here, we discover YAP/TAZ as bona fide downstream effectors of the alternative Wnt signaling pathway. Wnt5a/b and Wnt3a induce YAP/TAZ activation independent of canonical Wnt/ß-catenin signaling. Mechanistically, we delineate the "alternative Wnt-YAP/TAZ signaling axis" that consists of Wnt-FZD/ROR-Gα12/13-Rho GTPases-Lats1/2 to promote YAP/TAZ activation and TEAD-mediated transcription. YAP/TAZ mediate the biological functions of alternative Wnt signaling, including gene expression, osteogenic differentiation, cell migration, and antagonism of Wnt/ß-catenin signaling. Together, our work establishes YAP/TAZ as critical mediators of alternative Wnt signaling.

RAP2 mediates mechanoresponses of the Hippo pathway.

Mammalian cells are surrounded by neighbouring cells and extracellular matrix (ECM), which provide cells with structural support and mechanical cues that influence diverse biological processes1. The Hippo pathway effectors YAP (also known as YAP1) and TAZ (also known as WWTR1) are regulated by mechanical cues and mediate cellular responses to ECM stiffness2,3. Here we identified the Ras-related GTPase RAP2 as a key intracellular signal transducer that relays ECM rigidity signals to control mechanosensitive cellular activities through YAP and TAZ. RAP2 is activated by low ECM stiffness, and deletion of RAP2 blocks the regulation of YAP and TAZ by stiffness signals and promotes aberrant cell growth. Mechanistically, matrix stiffness acts through phospholipase Cγ1 (PLCγ1) to influence levels of phosphatidylinositol 4,5-bisphosphate and phosphatidic acid, which activates RAP2 through PDZGEF1 and PDZGEF2 (also known as RAPGEF2 and RAPGEF6). At low stiffness, active RAP2 binds to and stimulates MAP4K4, MAP4K6, MAP4K7 and ARHGAP29, resulting in activation of LATS1 and LATS2 and inhibition of YAP and TAZ. RAP2, YAP and TAZ have pivotal roles in mechanoregulated transcription, as deletion of YAP and TAZ abolishes the ECM stiffness-responsive transcriptome. Our findings show that RAP2 is a molecular switch in mechanotransduction, thereby defining a mechanosignalling pathway from ECM stiffness to the nucleus.

Characterization of Hippo Pathway Components by Gene Inactivation.

The Hippo pathway is important for regulating tissue homeostasis, and its dysregulation has been implicated in human cancer. However, it is not well understood how the Hippo pathway becomes dysregulated because few mutations in core Hippo pathway components have been identified. Therefore, much work in the Hippo field has focused on identifying upstream regulators, and a complex Hippo interactome has been identified. Nevertheless, it is not always clear which components are the most physiologically relevant in regulating YAP/TAZ. To provide an overview of important Hippo pathway components, we created knockout cell lines for many of these components and compared their relative contributions to YAP/TAZ regulation in response to a wide range of physiological signals. By this approach, we provide an overview of the functional importance of many Hippo pathway components and demonstrate NF2 and RHOA as important regulators of YAP/TAZ and TAOK1/3 as direct kinases for LATS1/2.

Regulation of the Hippo Pathway Transcription Factor TEAD.

The TEAD transcription factor family is best known for transcriptional output of the Hippo signaling pathway and has been implicated in processes such as development, cell growth and proliferation, tissue homeostasis, and regeneration. Our understanding of the functional importance of TEADs has increased dramatically since its initial discovery three decades ago. The majority of our knowledge of TEADs is in the context of Hippo signaling as nuclear DNA-binding proteins passively activated by Yes-associated protein (YAP) and transcriptional activator with PDZ-binding domain (TAZ), transcription coactivators downstream of the Hippo pathway. However, recent studies suggest that TEAD itself is actively regulated. Here, we highlight evidence demonstrating Hippo-independent regulation of TEADs and the potential impacts these studies may have on new cancer therapeutics.

A tiling-deletion-based genetic screen for cis-regulatory element identification in mammalian cells.

Millions of cis-regulatory elements are predicted to be present in the human genome, but direct evidence for their biological function is scarce. Here we report a high-throughput method, cis-regulatory element scan by tiling-deletion and sequencing (CREST-seq), for the unbiased discovery and functional assessment of cis-regulatory sequences in the genome. We used it to interrogate the 2-Mb POU5F1 locus in human embryonic stem cells, and identified 45 cis-regulatory elements. A majority of these elements have active chromatin marks, DNase hypersensitivity, and occupancy by multiple transcription factors, which confirms the utility of chromatin signatures in cis-element mapping. Notably, 17 of them are previously annotated promoters of functionally unrelated genes, and like typical enhancers, they form extensive spatial contacts with the POU5F1 promoter. These results point to the commonality of enhancer-like promoters in the human genome.

The Hippo pathway effector proteins YAP and TAZ have both distinct and overlapping functions in the cell.

The Hippo pathway plays an important role in regulating tissue homeostasis, and its effectors, the transcriptional co-activators Yes-associated protein (YAP) and WW domain-containing transcription regulator 1 (WWTR1 or TAZ), are responsible for mediating the vast majority of its physiological functions. Although YAP and TAZ are thought to be largely redundant and similarly regulated by Hippo signaling, they have developmental, structural, and physiological differences that suggest they may differ in their regulation and downstream functions. To better understand the functions of YAP and TAZ in the Hippo pathway, using CRISPR/Cas9, we generated YAP KO, TAZ KO, and YAP/TAZ KO cell lines in HEK293A cells. We evaluated them in response to many environmental conditions and stimuli and used RNA-Seq to compare their transcriptional profiles. We found that YAP inactivation has a greater effect on cellular physiology (namely, cell spreading, volume, granularity, glucose uptake, proliferation, and migration) than TAZ inactivation. However, functional redundancy between YAP and TAZ was also observed. In summary, our findings confirm that the Hippo pathway effectors YAP and TAZ are master regulators for multiple cellular processes but also reveal that YAP has a stronger influence than TAZ.

Heterogeneous resistance to quizartinib in acute myeloid leukemia revealed by single-cell analysis.

Genomic studies have revealed significant branching heterogeneity in cancer. Studies of resistance to tyrosine kinase inhibitor therapy have not fully reflected this heterogeneity because resistance in individual patients has been ascribed to largely mutually exclusive on-target or off-target mechanisms in which tumors either retain dependency on the target oncogene or subvert it through a parallel pathway. Using targeted sequencing from single cells and colonies from patient samples, we demonstrate tremendous clonal diversity in the majority of acute myeloid leukemia (AML) patients with activating FLT3 internal tandem duplication mutations at the time of acquired resistance to the FLT3 inhibitor quizartinib. These findings establish that clinical resistance to quizartinib is highly complex and reflects the underlying clonal heterogeneity of AML.

Crenolanib is a selective type I pan-FLT3 inhibitor.

Tyrosine kinase inhibitors (TKIs) represent transformative therapies for several malignancies. Two critical features necessary for maximizing TKI tolerability and response duration are kinase selectivity and invulnerability to resistance-conferring kinase domain (KD) mutations in the intended target. No prior TKI has demonstrated both of these properties. Aiming to maximize selectivity, medicinal chemists have largely sought to create TKIs that bind to an inactive (type II) kinase conformation. Here we demonstrate that the investigational type I TKI crenolanib is a potent inhibitor of Fms tyrosine kinase-3 (FLT3) internal tandem duplication, a validated therapeutic target in human acute myeloid leukemia (AML), as well as all secondary KD mutants previously shown to confer resistance to the first highly active FLT3 TKI quizartinib. Moreover, crenolanib is highly selective for FLT3 relative to the closely related protein tyrosine kinase KIT, demonstrating that simultaneous FLT3/KIT inhibition, a prominent feature of other clinically active FLT3 TKIs, is not required for AML cell cytotoxicity in vitro and may contribute to undesirable toxicity in patients. A saturation mutagenesis screen of FLT3-internal tandem duplication failed to recover any resistant colonies in the presence of a crenolanib concentration well below what has been safely achieved in humans, suggesting that crenolanib has the potential to suppress KD mutation-mediated clinical resistance. Crenolanib represents the first TKI to exhibit both kinase selectivity and invulnerability to resistance-conferring KD mutations, which is unexpected of a type I inhibitor. Crenolanib has significant promise for achieving deep and durable responses in FLT3-mutant AML, and may have a profound impact upon future medicinal chemistry efforts in oncology.

Activity of ponatinib against clinically-relevant AC220-resistant kinase domain mutants of FLT3-ITD.

Secondary point mutations in the Fms-like tyrosine kinase 3 (FLT3) tyrosine kinase domain (KD) are common causes of acquired clinical resistance to the FLT3 inhibitors AC220 (quizartinib) and sorafenib. Ponatinib (AP24534) is a multikinase inhibitor with in vitro and clinical activity in tyrosine kinase inhibitor (TKI)-resistant chronic myeloid leukemia, irrespective of BCR-ABL KD mutation. Ponatinib has demonstrated early clinical efficacy in chemotherapy-resistant acute myeloid leukemia (AML) patients with internal tandem duplication (ITD) mutations in FLT3. We assessed the in vitro activity of ponatinib against clinically relevant FLT3-ITD mutant isoforms that confer resistance to AC220 or sorafenib. Substitution of the FLT3 "gatekeeper" phenylalanine with leucine (F691L) conferred mild resistance to ponatinib, but substitutions at the FLT3 activation loop (AL) residue D835 conferred a high degree of resistance. Saturation mutagenesis of FLT3-ITD exclusively identified FLT3 AL mutations at positions D835, D839, and Y842. The switch control inhibitor DCC-2036 was similarly inactive against FLT3 AL mutations. On the basis of its in vitro activity against FLT3 TKI-resistant F691 substitutions, further clinical evaluation of ponatinib in TKI-naïve and select TKI-resistant FLT3-ITD+ AML patients is warranted. Alternative strategies will be required for patients with TKI-resistant FLT3-ITD D835 mutations.

The Irreversible FLT3 Inhibitor FF-10101 Is Active Against a Diversity of FLT3 Inhibitor Resistance Mechanisms.

Small-molecule FLT3 inhibitors have recently improved clinical outcomes for patients with FLT3-mutant acute myeloid leukemia (AML) after many years of development, but resistance remains an important clinical problem. FF-10101 is the first irreversible, covalent inhibitor of FLT3 which has previously shown activity against FLT3 tyrosine kinase inhibitor resistance-causing FLT3 F691L and D835 mutations. We report that FF-10101 is also active against an expanded panel of clinically identified FLT3 mutations associated with resistance to other FLT3 inhibitors. We also demonstrate that FF-10101 can potentially address resistance mechanisms associated with growth factors present in the bone marrow microenvironment but is vulnerable to mutation at C695, the amino acid required for covalent FLT3 binding. These data suggest that FF-10101 possesses a favorable resistance profile that may contribute to improved single-agent efficacy when used in patients with FLT3-mutant AML.

The Hippo pathway mediates Semaphorin signaling.

Semaphorins were originally identified as axonal guidance molecules, but they also control processes such as vascular development and tumorigenesis. The downstream signaling cascades of Semaphorins in these biological processes remain unclear. Here, we show that the class 3 Semaphorins (SEMA3s) activate the Hippo pathway to attenuate tissue growth, angiogenesis, and tumorigenesis. SEMA3B restoration in lung cancer cells with SEMA3B loss of heterozygosity suppresses cancer cell growth via activating the core Hippo kinases LATS1/2 (large tumor suppressor kinase 1/2). Furthermore, SEMA3 also acts through LATS1/2 to inhibit angiogenesis. We identified p190RhoGAPs as essential partners of the SEMA3A receptor PlexinA in Hippo regulation. Upon SEMA3 treatment, PlexinA interacts with the pseudo-guanosine triphosphatase (GTPase) domain of p190RhoGAP and simultaneously recruits RND GTPases to activate p190RhoGAP, which then stimulates LATS1/2. Disease-associated etiological factors, such as genetic lesions and oscillatory shear, diminish Hippo pathway regulation by SEMA3. Our study thus discovers a critical role of Hippo signaling in mediating SEMA3 physiological function.

Heat stress activates YAP/TAZ to induce the heat shock transcriptome.

The Hippo pathway plays critical roles in cell growth, differentiation, organ development and tissue homeostasis, whereas its dysregulation can lead to tumorigenesis. YAP and TAZ are transcription co-activators and represent the main downstream effectors of the Hippo pathway. Here, we show that heat stress induces a strong and rapid YAP dephosphorylation and activation. The effect of heat shock on YAP is dominant to other signals known to modulate the Hippo pathway. Heat shock inhibits LATS kinase by promoting HSP90-dependent LATS interaction with and inactivation by protein phosphatase 5. Heat shock also induces LATS ubiquitination and degradation. YAP and TAZ are crucial for cellular heat shock responses, including the heat shock transcriptome and cell viability. This study uncovers previously unknown mechanisms of Hippo regulation by heat shock, as well as physiological functions of YAP, in the heat stress response. Our observations also reveal a potential combinational therapy involving hyperthermia and targeting of the Hippo pathway.

Author Correction: Regulation of Hippo pathway transcription factor TEAD by p38 MAPK-induced cytoplasmic translocation.

In this Letter, the authors neglected to acknowledge funding from the Yonsei University Future-leading Research Initiative of 2017 (2017-22-007) awarded to H.W.P.

Regulation of Hippo pathway transcription factor TEAD by p38 MAPK-induced cytoplasmic translocation.

The Hippo pathway controls organ size and tissue homeostasis, with deregulation leading to cancer. The core Hippo components in mammals are composed of the upstream serine/threonine kinases Mst1/2, MAPK4Ks and Lats1/2. Inactivation of these upstream kinases leads to dephosphorylation, stabilization, nuclear translocation and thus activation of the major functional transducers of the Hippo pathway, YAP and its paralogue TAZ. YAP/TAZ are transcription co-activators that regulate gene expression primarily through interaction with the TEA domain DNA-binding family of transcription factors (TEAD). The current paradigm for regulation of this pathway centres on phosphorylation-dependent nucleocytoplasmic shuttling of YAP/TAZ through a complex network of upstream components. However, unlike other transcription factors, such as SMAD, NF-κB, NFAT and STAT, the regulation of TEAD nucleocytoplasmic shuttling has been largely overlooked. In the present study, we show that environmental stress promotes TEAD cytoplasmic translocation via p38 MAPK in a Hippo-independent manner. Importantly, stress-induced TEAD inhibition predominates YAP-activating signals and selectively suppresses YAP-driven cancer cell growth. Our data reveal a mechanism governing TEAD nucleocytoplasmic shuttling and show that TEAD localization is a critical determinant of Hippo signalling output.

Characterizing and Overriding the Structural Mechanism of the Quizartinib-Resistant FLT3 "Gatekeeper" F691L Mutation with PLX3397.

UNLABELLED: Tyrosine kinase domain mutations are a common cause of acquired clinical resistance to tyrosine kinase inhibitors (TKI) used to treat cancer, including the FLT3 inhibitor quizartinib. Mutation of kinase "gatekeeper" residues, which control access to an allosteric pocket adjacent to the ATP-binding site, has been frequently implicated in TKI resistance. The molecular underpinnings of gatekeeper mutation-mediated resistance are incompletely understood. We report the first cocrystal structure of FLT3 with the TKI quizartinib, which demonstrates that quizartinib binding relies on essential edge-to-face aromatic interactions with the gatekeeper F691 residue, and F830 within the highly conserved Asp-Phe-Gly motif in the activation loop. This reliance makes quizartinib critically vulnerable to gatekeeper and activation loop substitutions while minimizing the impact of mutations elsewhere. Moreover, we identify PLX3397, a novel FLT3 inhibitor that retains activity against the F691L mutant due to a binding mode that depends less vitally on specific interactions with the gatekeeper position. SIGNIFICANCE: We report the first cocrystal structure of FLT3 with a kinase inhibitor, elucidating the structural mechanism of resistance due to the gatekeeper F691L mutation. PLX3397 is a novel FLT3 inhibitor with in vitro activity against this mutation but is vulnerable to kinase domain mutations in the FLT3 activation loop.