Hippo Kinase Research Articles

Abstract 2294: NEK1 phosphorylation of YAP promotes its stabilization and transcriptional output

Abstract Background: Despite recent development in prostate cancer (PCa) therapeutics, castration-resistant prostate cancer still accounts for high morbidity and mortality in patients, partly because of incomplete knowledge of the major players that contribute to the androgen independent growth of prostate tumor. NIMA related kinase 1 (NEK1), a dual specificity kinase, mainly functions in maintaining genomic stability by regulating DNA damage response (DDR) and G2/M transition of the cells. We previously reported that tousled-like kinase 1 (TLK1) mediated phosphorylation of NEK1 on T141 can lead to rapid androgen independent progression of PCa cells upon anti-androgen treatment by activating DDR. However, activating DDR alone may not be sufficient to trigger androgen insensitive growth of PCa cells. We hypothesize that TLK1>NEK1 axis may also regulate hippo pathway oncoprotein YAP as a previous study reported that NEK1 can phosphorylate YAP homolog TAZ and upregulate YAP/TAZ transcriptional targets. Yes-associated protein (YAP) is a transcriptional co-activator and a major effector of hippo kinases that binds to transcription factor TEAD and promotes many oncogenic activities in the cells. Methods and Results: To determine if NEK1 can regulate Hippo pathway, our western blotting (WB) analysis revealed higher cleaved YAP (cl-YAP) and lower total YAP level in the NEK1-T141A hypoactive variant overexpressing LNCaP than wt-NEK1 overexpressing cells treated with bicalutamide (anti-androgen) and/or THD (a specific inhibitor of TLK1) which suggest that NEK1 may play a role in stabilizing YAP. TLK1 inhibition also resulted in the reduced expression of some YAP target genes such as CTGF, CDH2, Twist1, and P53AIP1. Co-IP assay revealed that NEK1 can interact with YAP regardless of its kinase activity. We generated a CRISPR-Cas9 mediated NEK1 knockout NT1 (a mouse PCa cell line) cells which showed reduced level of YAP in NEK1 KO clones as well as downregulation of YAP target genes such as CTGF, Zeb1, Twist1, and Cyr61. TLK1 knockdown and inhibition also resulted in reduced pNEK1 (T141) and total YAP level which suggested TLK1 mediated NEK1 activation is necessary for YAP stabilization. An IVK assay using [γ-32P] ATP demonstrated that catalytically active purified NEK1 can phosphorylate purified YAP1. MS analysis determined eight NEK1 mediated phospho-acceptor sites of YAP1: T83, T361, S366, S388, Y407, and T493. Y407 and T493 are the first ever reported phosphorylation sites on YAP1 especially by NEK1 which lies in the transactivation and PDZ binding domain. Our bioinformatics analysis revealed mRNA downregulation of YAP1, while upregulation of YAP protein level was observed in high-grade PCa, which is consistent with our model of post-transcriptional protein stabilization. Conclusion: Our data support that TLK1>NEK1 signaling promotes YAP stabilization and may contribute to castration resistant growth of PCa during AR blockade. Citation Format: Md Imtiaz Khalil, Ishita Ghosh, Vibha Singh, Jing Chen, Haining Zhu, Arrigo De Benedetti. NEK1 phosphorylation of YAP promotes its stabilization and transcriptional output [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2294.

Transcriptional Repression of RASSF2 Impairs Hippo Kinase-Dependent p53 Activation and Tumor Suppression in t(8;21) AML

The t(8;21) chromosomal translocation is a recurring cytogenetic abnormality in acute myeloid leukemia (AML), found in ~10% of AML patients. This translocation results in the stable fusion of the RUNX1 (AML1) and RUNX1T1 genes, and formation of the oncofusion protein RUNX1-ETO (RE). One function of RE in promoting leukemia development is the recruitment of aberrant transcription factor complexes to regulatory regions of RUNX1 target genes known to be critical for myeloid differentiation, such as CEBPA, SPI1, NFE2, and CSF1R . Despite this knowledge, additional RE target genes remain poorly characterized, and the complete molecular mechanism through which RE leads to leukemic transformation remains to be elucidated.Previously, we used a mouse model of t(8;21) AML to compare gene expression in RE-expressing leukemia cells to the normal mouse hematopoietic progenitor (lin-cKit+) compartment (Lo et al, 2012). Among the most significantly downregulated genes was Ras-association domain family member 2 (Rassf2). RASSF genes are frequently epigenetically silenced in human tumors, making them highly relevant to study in the context of cancer development. Using public gene expression data, RT-qPCR, and western blotting, we found that RASSF2 expression is specifically downregulated in t(8;21) AML cells compared to non-t(8;21) AML cells and normal CD34+ controls. Furthermore, RE knockdown in a t(8;21) AML cell line led to marked upregulation of RASSF2 mRNA. Based on ChiP-seq data analysis this repression appears to be mediated by direct RE binding and HDAC recruitment to the promoter region of both alternative RASSF2 transcriptional start sites in the genomic locus.To investigate the significance of this repression we used retroviral vectors to re-express RASSF2 in t(8;21) AML cell lines and RE-transduced primary murine hematopoietic cells in vivo and in vitro . Re-expression of RASSF2 significantly improved survival and reduced the total leukemic burden in a primary mouse model of RE-driven AML in vivo . RASSF2 inhibited leukemic transformation and the self-renewal ability of RE-transduced mouse bone marrow cells in a serial replating/colony formation assay, without a noticeable effect on normal hematopoietic progenitors. This inhibition correlated with increased apoptotic cells in the context of RE and RASSF2 co-expression.Given that RASSF2 is a non-enzymatic scaffolding protein, we hypothesized that it mediates tumor suppressive functions via its Ras-association or SARAH effector domains. We saw no detectable effect of RASSF2 expression on oncogenic Ras signaling in t(8;21) AML cells. This led us to investigate the SARAH domain-mediated interaction of RASSF2 with the pro-apoptotic Hippo kinases, MST1 and MST2. RASSF2 expression in t(8;21) AML cells led to an increased protein amount of both MST1 and MST2 in a SARAH domain-dependent manner. Importantly, use of either a RASSF2 SARAH domain deletion mutant, or Mst1(-/-)Mst2(-/-) mouse bone marrow cells, completely abrogated the ability of RASSF2 to inhibit RE leukemia cell self-renewal in the serial replating/colony formation assay. We found this inhibition to be independent of canonical Hippo pathway signaling, and therefore utilized a proximity-dependent biotin identification approach (BioID, Roux et al, 2012) to map the protein interactome of RASSF2-MST1/2 in a t(8;21) AML cell line. Interestingly, this revealed an interaction between RASSF2 and the protein deacetylase SIRT1. This highlighted a role for RASSF2 in promoting p53 acetylation in response to cellular stress, which is dependent on MST1/2-mediated negative regulation of SIRT1. Overall, our study revealed a critical tumor suppressive role of RASSF2-MST1/2 signaling via the SIRT1-p53 axis in t(8;21) AML. DisclosuresNo relevant conflicts of interest to declare.

LC3B phosphorylation regulates FYCO1 binding and directional transport of autophagosomes

Macroautophagy (hereafter referred to as autophagy) is a conserved process that promotes cellular homeostasis through the degradation of cytosolic components, also known as cargo. During autophagy, cargo is sequestered into double-membrane vesicles called autophagosomes, which are predominantly transported in the retrograde direction to the perinuclear region to fuse with lysosomes, thus ensuring cargo degradation.1 The mechanisms regulating directional autophagosomal transport remain unclear. The ATG8 family of proteins associates with autophagosome membranes2 and plays key roles in autophagy, including the movement of autophagosomes. This is achieved via the association of ATG8 with adaptor proteins like FYCO1, involved in the anterograde transport of autophagosomes toward the cell periphery.1,3-5 We previously reported that phosphorylation of LC3B/ATG8 on threonine 50 (LC3B-T50) by the Hippo kinase STK4/MST1 is required for autophagy through unknown mechanisms.6 Here, we show that STK4-mediated phosphorylation of LC3B-T50 reduces the binding of FYCO1 to LC3B. In turn, impairment of LC3B-T50 phosphorylation decreases starvation-induced perinuclear positioning of autophagosomes as well as their colocalization with lysosomes. Moreover, a significantly higher number of LC3B-T50A-positive autophagosomes undergo aberrant anterograde movement to axonal tips in mammalian neurons and toward the periphery of mammalian cells. Our data support a role of a nutrient-sensitive STK4-LC3B-FYCO1 axis in the regulation of the directional transport of autophagosomes, a key step of the autophagy process, via the post-translational modification of LC3B.

Dysregulation of the ESRP2-NF2-YAP/TAZ axis promotes hepatobiliary carcinogenesis in non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD), the hepatic correlate of the metabolic syndrome, is a major risk factor for hepatobiliary cancer (HBC). Although chronic inflammation is thought to be the root cause of all these diseases, the mechanism whereby it promotes HBC in NAFLD remains poorly understood. Herein, we aim to evaluate the hypothesis that inflammation-related dysregulation of the ESRP2-NF2-YAP/TAZ axis promotes HB carcinogenesis. We use murine NAFLD models, liver biopsies from patients with NAFLD, human liver cancer registry data, and studies in liver cancer cell lines. Our results confirm the hypothesis that inflammation-related dysregulation of the ESRP2-NF2-YAP/TAZ axis promotes HB carcinogenesis, supporting a model whereby chronic inflammation suppresses hepatocyte expression of ESRP2, an RNA splicing factor that directly targets and activates NF2, a tumor suppressor that is necessary to constrain YAP/TAZ activation. The resultant loss of NF2 function permits sustained YAP/TAZ activity that drives hepatocyte proliferation and de-differentiation. Herein, we report on a novel mechanism by which chronic inflammation leads to sustained activation of YAP/TAZ activity; this imposes a selection pressure that favors liver cells with mutations enabling survival during chronic oncogenic stress. Non-alcoholic fatty liver disease (NAFLD) increases the risk of hepatobiliary carcinogenesis. However, the underlying mechanism remains unknown. Our study demonstrates that chronic inflammation suppresses hepatocyte expression of ESRP2, an adult RNA splicing factor that activates NF2. Thus, inactive (fetal) NF2 loses the ability to activate Hippo kinases, leading to the increased activity of downstream YAP/TAZ and promoting hepatobiliary carcinogenesis in chronically injured livers.

Misshapen Disruption Cooperates with RasV12 to Drive Tumorigenesis.

Although RAS family genes play essential roles in tumorigenesis, effective treatments targeting RAS-related tumors are lacking, partly because of an incomplete understanding of the complex signaling crosstalk within RAS-related tumors. Here, we performed a large-scale genetic screen in Drosophila eye imaginal discs and identified Misshapen (Msn) as a tumor suppressor that synergizes with oncogenic Ras (RasV12) to induce c-Jun N-terminal kinase (JNK) activation and Hippo inactivation, then subsequently leads to tumor overgrowth and invasion. Moreover, ectopic Msn expression activates Hippo signaling pathway and suppresses Hippo signaling disruption-induced overgrowth. Importantly, we further found that Msn acts downstream of protocadherin Fat (Ft) to regulate Hippo signaling. Finally, we identified msn as a Yki/Sd target gene that regulates Hippo pathway in a negative feedback manner. Together, our findings identified Msn as a tumor suppressor and provide a novel insight into RAS-related tumorigenesis that may be relevant to human cancer biology.

Hippo-Independent Regulation of Yki/Yap/Taz: A Non-canonical View.

Initially identified in Drosophila, the Hippo signaling pathway has emerged as an evolutionarily conserved tumor suppressor pathway that controls tissue growth and organ size by simultaneously inhibiting cell proliferation and promoting cell death. Deregulation of Hippo pathway activity has been implicated in a wide range of human cancers. The core Hippo pathway consists of a kinase cascade: an upstream kinase Hippo (Hpo)/MST1/2 phosphorylates and activates a downstream kinase Warts (Wts)/Lats1/2, leading to phosphorylation and inactivation of a transcriptional coactivator Yki/YAP/Taz. Many upstream signals, including cell adhesion, polarity, mechanical stress, and soluble factors, regulate Hippo signaling through the kinase cascade, leading to change in the cytoplasmic/nuclear localization of Yki/YAP/Taz. However, recent studies have uncovered other mechanisms that regulate Yki/YAP/Taz subcellular localization, stability, and activity independent of the Hpo kinase cascade. These mechanisms provide additional layers of pathway regulation, nodes for pathway crosstalk, and opportunities for pathway intervention in cancer treatment and regenerative medicine.

MicroRNAs Regulating Hippo-YAP Signaling in Liver Cancer.

Liver cancer is one of the most common cancers worldwide, and its prevalence and mortality rate are increasing due to the lack of biomarkers and effective treatments. The Hippo signaling pathway has long been known to control liver size, and genetic depletion of Hippo kinases leads to liver cancer in mice through activation of the downstream effectors yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). Both YAP and TAZ not only reprogram tumor cells but also alter the tumor microenvironment to exert carcinogenic effects. Therefore, understanding the mechanisms of YAP/TAZ-mediated liver tumorigenesis will help overcome liver cancer. For decades, small noncoding RNAs, microRNAs (miRNAs), have been reported to play critical roles in the pathogenesis of many cancers, including liver cancer. However, the interactions between miRNAs and Hippo-YAP/TAZ signaling in the liver are still largely unknown. Here, we review miRNAs that influence the proliferation, migration and apoptosis of tumor cells by modulating Hippo-YAP/TAZ signaling during hepatic tumorigenesis. Previous findings suggest that these miRNAs are potential biomarkers and therapeutic targets for the diagnosis, prognosis, and treatment of liver cancer.

Inactivation of Lats1 and Lats2 highlights the role of hippo pathway effector YAP in larynx and vocal fold epithelium morphogenesis

Proliferation and differentiation of vocal fold epithelial cells during embryonic development is poorly understood. We examined the role of Hippo signaling, a vital pathway known for regulating organ size, in murine laryngeal development. Conditional inactivation of the Hippo kinase genes Lats1 and Lats2, specifically in vocal fold epithelial cells, resulted in severe morphogenetic defects. Deletion of Lats1 and Lats2 caused abnormalities in epithelial differentiation, epithelial lamina separation, cellular adhesion, basement membrane organization with secondary failed cartilage, and laryngeal muscle development. Further, Lats1 and Lats2 inactivation led to failure in differentiation of p63+ basal progenitors. Our results reveal novel roles of Hippo-Lats-YAP signaling in proper regulation of VF epithelial fate and larynx morphogenesis.

Astrocytic YAP protects the optic nerve and retina in an experimental autoimmune encephalomyelitis model through TGF-β signaling.

Rationale: Optic neuritis is one of main symptoms in multiple sclerosis (MS) that causes visual disability. Astrocytes are pivotal regulators of neuroinflammation in MS, and astrocytic yes-associated protein (YAP) plays a critical role in neuroinflammation. Meanwhile, YAP signaling is involved in visual impairment, including glaucoma, retinal choroidal atrophy and retinal detachment. However, the roles and underlying mechanisms of astrocytic YAP in neuroinflammation and demyelination of MS-related optic neuritis (MS-ON) remains unclear.Methods: To assess the functions of YAP in MS-ON, experimental autoimmune encephalomyelitis (EAE, a common model of MS) was established, and mice that conditional knockout (CKO) of YAP in astrocytes, YAPGFAP-CKO mice, were successfully generated. Behavior tests, immunostaining, Nissl staining, Hematoxylin-Eosin (HE) staining, TUNEL staining, Luxol Fast Blue (LFB) staining, electron microscopy (EM), quantitative real-time PCR (qPCR), gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) by RNA sequencing were used to examine the function and mechanism of YAP signaling based on these YAPGFAP-CKO mice and EAE model mice. To further explore the potential treatment of YAP signaling in EAE, EAE mice were treated with various drugs, including SRI-011381 that is an agonist of transforming growth factor-β (TGF-β) pathway, and XMU-MP-1 which inhibits Hippo kinase MST1/2 to activate YAP.Results: We found that YAP was significantly upregulated and activated in the astrocytes of optic nerve in EAE mice. Conditional knockout of YAP in astrocytes caused more severe inflammatory infiltration and demyelination in optic nerve, and damage of retinal ganglion cells (RGCs) in EAE mice. Moreover, YAP deletion in astrocytes promoted the activation of astrocytes and microglia, but inhibited the proliferation of astrocytes of optic nerve in EAE mice. Mechanically, TGF-β signaling pathway was significantly down-regulated after YAP deletion in astrocytes. Additionally, both qPCR and immunofluorescence assays confirmed the reduction of TGF-β signaling pathway in YAPGFAP-CKO EAE mice. Interestingly, SRI-011381 partially rescued the deficits in optic nerve and retina of YAPGFAP-CKO EAE mice. Finally, activation of YAP signaling by XMU-MP-1 relieved the neuroinflammation and demyelination in optic nerve of EAE mice.Conclusions: These results suggest astrocytic YAP may prevent the neuroinflammatory infiltration and demyelination through upregulation of TGF-β signaling and provide targets for the development of therapeutic strategies tailored for MS-ON.

Lipid-Raft-Targeted Molecular Self-Assembly Inactivates YAP to Treat Ovarian Cancer.

The Yes-associated protein (YAP) is a major oncoprotein responsible for cell proliferation control. YAP's oncogenic activity is regulated by both the Hippo kinase cascade and uniquely by a mechanical-force-induced actin remodeling process. Inspired by reports that ovarian cancer cells specifically accumulate the phosphatase protein ALPP on lipid rafts that physically link to actin cytoskeleton, we developed a molecular self-assembly (MSA) technology that selectively halts cancer cell proliferation by inactivating YAP. We designed a ruthenium-complex-peptide precursor molecule that, upon cleavage of phosphate groups, undergoes self-assembly to form nanostructures specifically on lipid rafts of ovarian cancer cells. The MSAs exert potent, cancer-cell-specific antiproliferative effects in multiple cancer cell lines and in mouse xenograft tumor models. Our work illustrates how basic biochemical insights can be exploited as the basis for a nanobiointerface fabrication technology which links nanoscale protein activities at specific subcellular locations to molecular biological activities to suppress cancer cell proliferation.

Modification of COL1A1 in Autologous Adipose Tissue-Derived Progenitor Cells Rescues the Bone Phenotype in a Mouse Model of Osteogenesis Imperfecta.

Osteogenesis imperfecta (OI) is a congenital genetic disorder mainly manifested as bone fragility and recurrent fracture. Mutation of COL1A1/COL1A2 genes encoding the type I collagen are most responsible for the clinical patients. Allogenic mesenchymal stem cells (MSCs) provide the potential to treat OI through differentiation into osteoblasts. Autologous defective MSCs have not been utilized in OI treatment mainly because of their impaired osteogenesis, but the latent mechanism has not been well understood. Here, the relative signaling abnormality of adipose-derived mesenchymal stem cells (ADSCs) isolated from OI type I mice (Col1a1+/-365 mice) was explored. Autologous ADSCs transfected by retrovirus carrying human COL1A1 gene was first utilized in OI therapy. The results showed that decreased activity of Yes-associated protein (YAP) due to hyperactive upstream Hippo kinases greatly contributed to the weakened bone-forming capacity of defective ADSCs. Recovered collagen synthesis of autologous ADSCs by COL1A1 gene modification normalized Hippo/YAP signaling and effectively rescued YAP-mediated osteogenesis. And the COL1A1 gene engineered autologous ADSCs efficaciously improved the microstructure, enhanced the mechanical properties and promoted bone formation of Col1a1+/-365 mice after femoral bone marrow cavity delivery and might serve as an alternative source of stem cells in OI treatment. © 2021 American Society for Bone and Mineral Research (ASBMR).

Hepatic Hippo signaling inhibits development of hepatocellular carcinoma.

Primary liver cancer is one of the most common cancer worldwide. Hepatocellular carcinoma (HCC) in particular, is the second leading cause of cancer deaths in the world. The Hippo signaling pathway has emerged as a major oncosuppressive pathway that plays critical roles inhibiting hepatocyte proliferation, survival, and HCC formation. A key component of the Hippo pathway is the inhibition of yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ) transcription factors by the Hippo kinase cascade. Aberrant activation of YAP or TAZ has been found in several human cancers including HCC. It is also well established that YAP/TAZ activation in hepatocytes causes HCC in mouse models, indicating that YAP/TAZ are potential therapeutic targets for human liver cancer. In this review, we summarize the recent findings regarding the multifarious roles of Hippo/YAP/TAZ in HCC development, and focus on their cell autonomous roles in controlling hepatocyte proliferation, differentiation, survival and metabolism as well as their non-cell autonomous in shaping the tumor microenvironment.

Centrosome Reduction Promotes Terminal Differentiation of Human Cardiomyocytes.

SummaryCentrosome reduction and redistribution of pericentriolar material (PCM) coincides with cardiomyocyte transitions to a post-mitotic and matured state. However, it is unclear whether centrosome changes are a cause or consequence of terminal differentiation. We validated that centrosomes were intact and functional in proliferative human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), consistent with their immature phenotype. We generated acentrosomal hPSC-CMs, through pharmacological inhibition of centriole duplication, and showed that centrosome loss was sufficient to promote post-mitotic transitions and aspects of cardiomyocyte maturation. As Hippo kinases are activated during post-natal cardiac maturation, we pharmacologically activated the Hippo pathway using C19, which was sufficient to trigger centrosome disassembly and relocalization of PCM components to perinuclear membranes. This was due to specific activation of Hippo kinases, as direct inhibition of YAP-TEAD interactions with verteporfin had no effect on centrosome organization. This suggests that Hippo kinase-centrosome remodeling may play a direct role in cardiac maturation.

Abstract 124: Recycling endosomes in mature epithelia restrain tumorigenic signaling

Abstract The effects of polarized membrane trafficking in mature epithelial tissue on cell growth and cancer progression have not been fully explored in vivo. A majority of colorectal cancers have reduced and mislocalized Rab11, a small GTPase dedicated to trafficking of recycling endosomes. Patients with low Rab11 protein expression have poor survival rates. Using genetic models across species, we show that intact recycling endosome function restrains aberrant epithelial growth elicited by APC or RAS mutations. Loss of Rab11 protein led to epithelial dysplasia in early animal development and synergized with oncogenic pathways to accelerate tumor progression initiated by carcinogen, genetic mutation, or aging. Transcriptomic analysis uncovered an immediate expansion of the intestinal stem cell pool along with cell-autonomous Yki/Yap activation following disruption of Rab11a-mediated recycling endosomes. Intestinal tumors lacking Rab11a traffic exhibited marked elevation of nuclear Yap, upd3/IL6-Stat3, and amphiregulin-MAPK signaling, while suppression of Yki/Yap or upd3/IL6 reduced gut epithelial dysplasia and hyperplasia. Examination of Rab11a function in enteroids or cultured cell lines suggested that this endosome unit is required for suppression of the Yap pathway by Hippo kinases. Thus, recycling endosomes in mature epithelia constitute key tumor suppressors, loss of which accelerates carcinogenesis. Statement of significance: Recycling endosome traffic in mature epithelia constitutes a novel tumor suppressing mechanism. Citation Format: Sayantani Goswami. Recycling endosomes in mature epithelia restrain tumorigenic signaling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 124.

Abstract B12: High-throughput screening platform to discover TEAD modulators

Abstract The TEAD family of transcription factors play critical roles during development, cell growth, proliferation, regeneration, and tissue homeostasis. TEADs are key downstream transcription factors of the evolutionarily conserved Hippo pathway, which binds Yes-associated protein (YAP) and transcriptional activator with PDZ-binding domain (TAZ) transcriptional coactivators through their transactivation domains. Despite TEAD being the key transducer of the Hippo signaling, most research have been focused on Hippo kinases and YAP/TAZ, whereas the Hippo-dependent and -independent regulators and regulations governing TEAD emerged only recently. Previously, we identified that environmental stress promotes TEAD cytoplasmic translocation via p38 MAPK-dependent, Hippo-independent mechanism, which overrides YAP-driven oncogenic growth in cancer cells. Here, we present a platform for high-throughput screening to identify chemical compounds that modulate TEAD cellular localization and activity. We designed cancer cells stably expressing a TEAD-GFP lentiviral vector and carried out a comprehensive comparative analysis of TEAD4 by GFP-fluorescence, immunoblotting, and transcript activities. Chemical compound screening to discover TEAD modulators will reveal novel anticancer drugs. Citation Format: Sungho Moon, Hyun Woo Park. High-throughput screening platform to discover TEAD modulators [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B12.

Abstract IA16: Regulation of SAV1-STRIPAK antagonism by YSK1-dependent phosphorylation

Abstract The Hippo pathway is critical for tissue homeostasis and tumor suppression in multicellular organisms. Through cell-surface receptors and cytoskeletal complexes, extracellular signals, including cell-cell contact, activate a core kinase cascade formed by the Hippo kinases MST1/2, the NDR family kinases LATS1/2, and the adaptor proteins SAV1 and MOB1. Activated LATS1/2 phosphorylate and prevent the nuclear accumulation of the transcription factors YAP/TAZ. Activation of MST1/2 initiates Hippo signaling and requires trans-autophosphorylation in the T-loop (MST2 T180). We have recently shown that the PP2A complex STRIPAK blocks MST2 activation through feedback inhibition. SAV1 promotes MST2 activation by suppressing STRIPAKSLMAP-mediated dephosphorylation of MST2 pT180. Our preliminary results show that YSK1 (the kinase component of STRIPAK) phosphorylates SAV1 and blocks its ability to inhibit PP2A. We hypothesize that upstream signals impinge on the SAV1-STRIPAK antagonism through YSK1 to regulate MST1/2 activation. Citation Format: Xuelian “Sue” Luo. Regulation of SAV1-STRIPAK antagonism by YSK1-dependent phosphorylation [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr IA16.

Abstract B06: Development of selective LATS1/LATS2 inhibitors for the pharmacologic modulation of the Hippo signaling pathway

Abstract We performed a large-scale profiling of currently available kinase inhibitors (KI) to identify “off-targets” that could be repurposed/repositioned as the starting point for the rational development of novel and selective kinase inhibitors against underdrugged targets. We profiled 257 KI at a single dose against a panel of 365 wild-type kinases tested individually, for a total of about 100,000 in vitro drug/kinase data points. After unsupervised hierarchical clustering of this dataset based on percent inhibition values, we identified a well-resolved subcluster driven by 5 KI with limited polypharmacology and a clean kinase-inhibition profile, mostly targeting AGC group kinases. These inhibitors were the AKT inhibitors GSK-690693, AZD-5363, and AT-7867; the MSK inhibitor SB-747651-A; and the Rho kinases (ROCK) inhibitor GSK-269962A. Notably, the AGC and hippo kinases LATS1 and LATS2 were part of this subcluster and were both inhibited to significant extents by these 5 compounds. With the regenerative potential of a candidate LATS1/2 inhibitor in mind, we next developed several orthogonal biochemical and cellular assays to prioritize compounds that possessed the desired activity against LATS1/2 without the burden of cytotoxic “off-targets,” such as those resulting from strong AKT inhibition. Based on the results of these assays, we prioritized one of the initially identified kinase inhibitors as the starting point for an ongoing, rational structure-activity relationship (SAR) effort to develop novel, 1st-in-class, highly selective LATS1 and LATS2 inhibitors. We will present data showing how such novel LATS1/2 inhibitors efficiently block LATS1/2-dependent YAP phosphorylation, induce YAP nuclear translocation in a high cell-density setting, and promote the transcription of bona fide YAP target genes such as AMOTL2, CTGF, and CYR61. Finally, consistently with the activation of a YAP-dependent transcriptional program, small-molecule inhibitors of LATS1/2 kinases promoted proliferation/regeneration in an in vitro wound-healing assay. Altogether, we identified novel small molecules that can be used as chemical probes to acutely inactivate the Hippo signaling pathway and validated LATS1 and LATS2 kinases as novel druggable kinases whose inhibition could be exploited for several indications, spanning from regenerative medicine to cancer immunotherapy. Citation Format: Michele Ceribelli, Patrick Morris, Damien Duveau, Frances A. Tosto, Scott Hoyt, Craig J. Thomas. Development of selective LATS1/LATS2 inhibitors for the pharmacologic modulation of the Hippo signaling pathway [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B06.

Regenerative Reprogramming of the Intestinal Stem Cell State via Hippo Signaling Suppresses Metastatic Colorectal Cancer.

Although the Hippo transcriptional coactivator YAP is considered oncogenic in many tissues, its roles in intestinal homeostasis and colorectal cancer (CRC) remain controversial. Here, we demonstrate that the Hippo kinases LATS1/2 and MST1/2, which inhibit YAP activity, are required for maintaining Wnt signaling and canonical stem cell function. Hippo inhibition induces a distinct epithelial cell state marked by low Wnt signaling, a wound-healing response, and transcription factor Klf6 expression. Notably, loss of LATS1/2 or overexpression of YAP is sufficient to reprogram Lgr5+ cancer stem cells to this state and thereby suppress tumor growth in organoids, patient-derived xenografts, and mouse models of primary and metastatic CRC. Finally, we demonstrate that genetic deletion of YAP and its paralog TAZ promotes the growth of these tumors. Collectively, our results establish the role of YAP as a tumor suppressor in the adult colon and implicate Hippo kinases as therapeutic vulnerabilities in colorectal malignancies.

Leukaemia Inhibitory Factor (LIF) Inhibits Cancer Stem Cells Tumorigenic Properties through Hippo Kinases Activation in Gastric Cancer.

Cancer stem cells (CSCs) present chemo-resistance mechanisms contributing to tumour maintenance and recurrence, making their targeting of utmost importance in gastric cancer (GC) therapy. The Hippo pathway has been implicated in gastric CSC properties and was shown to be regulated by leukaemia inhibitory factor receptor (LIFR) and its ligand LIF in breast cancer. This study aimed to determine LIF’s effect on CSC properties in GC cell lines and patient-derived xenograft (PDX) cells, which remains unexplored. LIF’s treatment effect on CSC markers expression and tumoursphere formation was evaluated. The Hippo kinase inhibitor XMU-MP-1 and/or the JAK1 inhibitor Ruxolitinib were used to determine Hippo and canonical JAK/STAT pathway involvement in gastric CSCs’ response to LIF. Results indicate that LIF decreased tumorigenic and chemo-resistant CSCs, in both GC cell lines and PDX cells. In addition, LIF increased activation of LATS1/2 Hippo kinases, thereby decreasing downstream YAP/TAZ nuclear accumulation and TEAD transcriptional activity. LIF’s anti-CSC effect was reversed by XMU-MP-1 but not by Ruxolitinib treatment, highlighting the opposite effects of these two pathways downstream LIFR. In conclusion, LIF displays anti-CSC properties in GC, through Hippo kinases activation, and could in fine constitute a new CSCs-targeting strategy to help decrease relapse cases and bad prognosis in GC.

Selective Inhibition of STRN3-Containing PP2A Phosphatase Restores Hippo Tumor-Suppressor Activity in Gastric Cancer

Loss of Hippo tumor-suppressor activity and hyperactivation of YAP are commonly observed in cancers. Inactivating mutations of Hippo kinases MST1/2 are uncommon, and it remains unclear how their activity is turned off during tumorigenesis. We identified STRN3 as an essential regulatory subunit of protein phosphatase 2A (PP2A) that recruits MST1/2 and promotes its dephosphorylation, which results in YAP activation. We also identified STRN3 upregulation in gastric cancer correlated with YAP activation and poor prognosis. Based on this mechanistic understanding and aided by structure-guided medicinal chemistry, we developed a highly selective peptide inhibitor, STRN3-derived Hippo-activating peptide, or SHAP, which disrupts the STRN3-PP2Aa interaction and reactivates the Hippo tumor suppressor, inhibits YAP activation, and has antitumor effects invivo.