NIBR-LTSi is a selective LATS kinase inhibitor activating YAP signaling and expanding tissue stem cells in vitro and in vivo.

The YAP/Hippo pathway is an organ growth and size regulation rheostat safeguarding multiple tissue stem cell compartments. LATS kinases phosphorylate and thereby inactivate YAP, thus representing a potential direct drug target for promoting tissue regeneration. Here, we report the identification and characterization of the selective small-molecule LATS kinase inhibitor NIBR-LTSi. NIBR-LTSi activates YAP signaling, shows good oral bioavailability, and expands organoids derived from several mouse and human tissues. In tissue stem cells, NIBR-LTSi promotes proliferation, maintains stemness, and blocks differentiation in vitro and in vivo. NIBR-LTSi accelerates liver regeneration following extended hepatectomy in mice. However, increased proliferation and cell dedifferentiation in multiple organs prevent prolonged systemic LATS inhibition, thus limiting potential therapeutic benefit. Together, we report a selective LATS kinase inhibitor agonizing YAP signaling and promoting tissue regeneration in vitro and in vivo, enabling future research on the regenerative potential of the YAP/Hippo pathway.

Inhibition of LATS kinases reduces tumorigenicity and increases the sensitivity of human chronic myelogenous leukemia cells to imatinib.

Chronic myelogenous leukemia (CML) is a clonal hematologic malignancy of the myeloid lineage caused by the oncogenic BCR/ABL fusion protein that promotes CML cell proliferation and protects them against drug-induced apoptosis. In this study, we determine LATS1 and LATS2 expression in CML cells derived from patients who are resistant to imatinib (IM) treatment. Significant upregulation of LATS1 and LATS2 was found in these CML patients compared to healthy donors. To further explore whether the expression of LATS1/2 contributes to the IM-resistant phenotype, IM-resistant CML cell lines generated by culturing CML-derived erythroblastic K562 cells in increasing concentrations of IM were used as in vitro models. Up-regulation of LATS1 and LATS2 was observed in IM-resistant K562 cells. Reduction of LATS using either Lats-IN-1 (TRULI), a specific LATS inhibitor, or shRNA targeting LATS1/2 significantly reduced clonogenicity, increased apoptosis and induced differentiation of K562 cells to late-stage erythroid cells. Furthermore, depletion of LATS1 and LATS2 also increased the sensitivity of K562 cells to IM. Taken together, our results suggest that LATS could be one of the key factors contributing to the rapid proliferation, reduced apoptosis, and IM resistance of CML cells. Targeting LATS could be a promising treatment to enhance the therapeutic effect of a conventional BCR/ABL tyrosine kinase inhibitor such as IM.

Efficient Reprogramming of Mouse Embryonic Stem Cells into Trophoblast Stem-like Cells via Lats Kinase Inhibition.

Mouse zygotes undergo multiple rounds of cell division, resulting in the formation of preimplantation blastocysts comprising three lineages: trophectoderm (TE), epiblast (EPI), and primitive endoderm (PrE). Cell fate determination plays a crucial role in establishing a healthy pregnancy. The initial separation of lineages gives rise to TE and inner cell mass (ICM), from which trophoblast stem cells (TSC) and embryonic stem cells (ESC) can be derived in vitro. Studying lineage differentiation is greatly facilitated by the clear functional distinction between TSC and ESC. However, transitioning between these two types of cells naturally poses challenges. In this study, we demonstrate that inhibiting LATS kinase promotes the conversion of ICM to TE and also effectively reprograms ESC into stable, self-renewing TS-like cells (TSLC). Compared to TSC, TSLC exhibits similar molecular properties, including the high expression of marker genes such as Cdx2, Eomes, and Tfap2c, as well as hypomethylation of their promoters. Importantly, TSLC not only displays the ability to differentiate into mature trophoblast cells in vitro but also participates in placenta formation in vivo. These findings highlight the efficient reprogramming of ESCs into TSLCs using a small molecular inducer, which provides a new reference for understanding the regulatory network between ESCs and TSCs.

The NDR/LATS protein kinases in neurobiology: Key regulators of cell proliferation, differentiation and migration in the ocular and central nervous system.

Nuclear Dbf2-related (NDR) kinases are a subgroup of evolutionarily conserved AGC protein kinases that regulate various aspects of cell growth and morphogenesis. There are 4 NDR protein kinases in mammals, LATS1, LATS2 and STTK8/NDR1, STK38L/NDR2 protein kinases. LATS1 and 2 are core components of the well-studied Hippo pathway, which play a critical role in the regulation of cell proliferation, differentiation, and cell migration via YAP/TAZ transcription factor. The Hippo pathways play an important role in nervous tissue development and homeostasis, especially with regard to the central nervous system (CNS) and the ocular system. The ocular system is a very complex system generated by the interaction in a very tightly coordinated manner of numerous and diverse developing tissues, such as, but not limited to choroidal and retinal blood vessels, the retinal pigmented epithelium and the retina, a highly polarized neuronal tissue. The retina development and maintenance require precise and coordinated regulation of cell proliferation, cell death, migration, morphogenesis, synaptic connectivity, and balanced homeostasis. This review highlights the emerging roles of NDR1 and NDR2 kinases in the regulation of retinal/neuronal function and homeostasis via a noncanonical branch of the Hippo pathway. We highlight a potential role of NDR1 and NDR2 kinases in regulating neuronal inflammation and as potential therapeutic targets for the treatment of neuronal diseases.

N-(3,4-dimethoxyphenethyl)-6-methyl-2,3,4,9-tetrahydro-1H-carbazol-1-amine inhibits bladder cancer progression by suppressing YAP1/TAZ.

Bladder cancer (BlC) is the fourth most common cancer in males worldwide, but few systemic chemotherapy options for its effective treatment exist. The development of new molecularly-targeted agents against BlC is therefore an urgent issue. The Hippo signaling pathway, with its upstream LATS kinases and downstream transcriptional co-activators YAP1 and TAZ, plays a pivotal role in diverse cell functions, including cell proliferation. Recent studies have shown that overexpression of YAP1 occurs in advanced BlCs and is associated with poor patient prognosis. Accessing data from our previous screening of a chemical library of compounds targeting the Hippo pathway, we identified DMPCA (N-(3,4-dimethoxyphenethyl)-6-methyl-2,3,4,9-tetrahydro-1H-carbazol-1-amine) as an agent able to induce the phosphorylation of LATS1 and YAP1/TAZ in BlC cells, thereby suppressing their viability both in vitro and in mouse xenografts. Our data indicate that DMPCA has a potent anti-tumor effect, and raise the possibility that this agent may represent a new and effective therapeutic option for BlC.

Fission Yeast NDR/LATS Kinase Orb6 Regulates Exocytosis via Phosphorylation of the Exocyst Complex.

NDR/LATS kinases regulate multiple aspects of cell polarity and morphogenesis from yeast to mammals. Fission yeast NDR/LATS kinase Orb6 has been proposed to control cell polarity by regulating the Cdc42 guanine nucleotide exchange factor Gef1. Here, we show that Orb6 regulates polarity largely independently of Gef1 and that Orb6 positively regulates exocytosis. Through Orb6 inhibition in vivo and quantitative global phosphoproteomics, we identify Orb6 targets, including proteins involved in membrane trafficking. We confirm Sec3 and Sec5, conserved components of the exocyst complex, as substrates of Orb6 both in vivo and in vitro, and we show that Orb6 kinase activity is important for exocyst localization to cell tips and for exocyst activity during septum dissolution after cytokinesis. We further find that Orb6 phosphorylation of Sec3 contributes to exocyst function in concert with exocyst protein Exo70. We propose that Orb6 contributes to polarized growth by regulating membrane trafficking at multiple levels.

Hippo signaling determines the number of venous pole cells that originate from the anterior lateral plate mesoderm in zebrafish.

The differentiation of the lateral plate mesoderm cells into heart field cells constitutes a critical step in the development of cardiac tissue and the genesis of functional cardiomyocytes. Hippo signaling controls cardiomyocyte proliferation, but the role of Hippo signaling during early cardiogenesis remains unclear. Here, we show that Hippo signaling regulates atrial cell number by specifying the developmental potential of cells within the anterior lateral plate mesoderm (ALPM), which are incorporated into the venous pole of the heart tube and ultimately into the atrium of the heart. We demonstrate that Hippo signaling acts through large tumor suppressor kinase 1/2 to modulate BMP signaling and the expression of hand2, a key transcription factor that is involved in the differentiation of atrial cardiomyocytes. Collectively, these results demonstrate that Hippo signaling defines venous pole cardiomyocyte number by modulating both the number and the identity of the ALPM cells that will populate the atrium of the heart.

Evolutionary and molecular facts link the WWC protein family to Hippo signaling.

The scaffolding protein KIBRA (also called WWC1) is involved in the regulation of important intracellular transport processes and the establishment of cell polarity. Furthermore, KIBRA/WWC1 is an upstream regulator of the Hippo signaling pathway that controls cell proliferation and organ size in animals. KIBRA/WWC1 represents only one member of the WWC protein family that also includes the highly similar proteins WWC2 and WWC3. Although the function of KIBRA/WWC1 was studied intensively in cells and animal models, the importance of WWC2 and WWC3 was not yet elucidated. Here, we describe evolutionary, molecular, and functional aspects of the WWC family. We show that the WWC genes arose in the ancestor of bilateral animals (clades such as insects and vertebrates) from a single founder gene most similar to the present KIBRA/WWC1-like sequence of Drosophila. This situation was still maintained until the common ancestor of lancelet and vertebrates. In fish, a progenitor-like sequence of mammalian KIBRA/WWC1 and WWC2 is expressed together with WWC3. Finally, in all tetrapods, the three family members, KIBRA/WWC1, WWC2, and WWC3, are found, except for a large genomic deletion including WWC3 in Mus musculus. At the molecular level, the highly conserved WWC proteins share a similar primary structure, the ability to form homo- and heterodimers and the interaction with a common set of binding proteins. Furthermore, all WWC proteins negatively regulate cell proliferation and organ growth due to a suppression of the transcriptional activity of YAP, the major effector of the Hippo pathway.

Actin-binding and cell proliferation activities of angiomotin family members are regulated by Hippo pathway-mediated phosphorylation.

Whether the Hippo pathway has downstream targets other than YAP and TAZ is unknown. In this report, we have identified angiomotin (Amot) family members as novel substrates of Hippo core kinases. The N-terminal regions of Amot proteins contain a conserved HXRXXS consensus site for LATS1/2-mediated phosphorylation. Phospho-specific antibodies showed that Hippo core kinases could mediate phosphorylation of endogenous as well as exogenous Amot family members. Knockdown of LATS1 and LATS2 endogenously reduced the phosphorylation of Amots detected by the phospho-specific antibodies. Mutation of the serine to alanine within this HXRXXS site in Amot and AmotL2 established that this site was essential for Hippo core kinase-mediated phosphorylation. Wild-type and non-phosphorylated Amot (Amot-S175A) were targeted to actin filaments, whereas phospho-mimic Amot (Amot-S175D) failed to be localized with actin. Overexpression of LATS2 caused dissociation of Amot from actin but not Amot-S175A. Mapping of the actin-binding site of Amot showed that serine 175 of Amot was important for the actin-binding activity. Amot-S175A promoted, whereas Amot and Amot-S175D inhibited, cell proliferation. These results collectively suggest that the Hippo pathway negatively regulates the actin-binding activity of Amot family members through direct phosphorylation.