bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A05: RAS signaling promotes ERMS cell viability via sustaining TAZ expression and protein stability
Authors: Lin Liz Yi-Tzu , Linardic Corinne
Published: September 2020

Rhabdomyosarcoma (RMS) is a pediatric cancer characterized by skeletal muscle histogenesis, with the embryonal histologic variant (ERMS) being the most common. Mutations of the RAS pathway are found in 40-60% of ERMS patient specimens, and as in other human cancers the RAS protein is currently not druggable, and inhibition of downstream RAS effector pathways fails due to compensating signaling. Thus, there is an urgent need to understand the dysregulated signaling in RAS-driven ERMS (rERMS). Hippo signaling was originally identified for its role in controlling organ size and tissue homeostasis, but it is now known to also have a critical role in cancer initiation and progression. In ERMS patients, higher YAP1 gene expression correlates with a poor prognosis, and in vitro and in vivo studies using ERMS cell lines and mouse models also show that YAP1 supports ERMS cell and tumor growth. Whether and how the YAP1 paralog WWTR1 (TAZ) contributes to rERMS tumorigenesis remains understudied. Our initial studies examining ERK suppression (genetic inhibition via shRNA or pharmacologic inhibition via trametinib) showed that this slowed rERMS cell growth and proliferation, and induced rERMS cell differentiation. However, we also found CTGF and CYR61 (downstream targets of YAP1/TAZ) significantly repressed upon trametinib treatment. While YAP1 appeared unaffected, trametinib treatment significantly suppressed TAZ at both the mRNA and protein levels in a dose-dependent manner. Suppression of TAZ with shRNA decreased rERMS cell growth and proliferation, suggesting that sustained TAZ expression is crucial for rERMS cell viability. Interestingly, there was no change in the activity of MST and LATS. Our next focus will be on the mechanism of ERK-mediated TAZ expression, with a potential role for glycogen synthase kinase 3 beta as a mediator of TAZ degradation. In summary, these studies identify a new role for RAS signaling in promoting rERMS cell survival via TAZ expression.

Citation Format: Liz Yi-Tzu Lin, Corinne Linardic. RAS signaling promotes ERMS cell viability via sustaining TAZ expression and protein stability [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 A05.
Abstract A10: Taz regulates aging of hematopoietic stem cells
Authors: Mura-Meszaros Anna , Heidel Florian , von Eyss Bjoern
Published: September 2020

During aging, several tissues display a progressive functional decline potentially leading to malfunction of whole organ systems. This phenomenon can also be observed in the hematopoietic system since aged hematopoietic stem cells (HSCs) display several cell-intrinsic defects, e.g., a strongly reduced self-renewal capacity as well as a skewing towards myeloid differentiation. To investigate a potential involvement of the Hippo pathway in the aging of HSCs, we performed RNA-sequencing in several stem and progenitor populations of young and aged mice, respectively. Here, we identified Taz as one of the most strongly upregulated genes, specifically in long-term hemtatopoietic stem cells (LT-HSCs), those cells that are at the apex of the hematopoietic system. Further analyses revealed that Taz actively contributes to the aging-specific gene expression changes in LT-HSCs and that Tazhigh cells accumulate over time in the LT-HSC compartment. Furthermore, we identified a novel surface marker as a Taz target gene, which allows us to trace Taz activity in the aging mouse and separate Tazhigh from Tazlow cells. Strikingly, our functional characterization revealed that Tazlow cells closely resemble young LT-HSCs whereas Tazhigh cells display a gene expression program that is found in aged LT-HSCs. Consistent with these observations, Tazhigh cells are functionally impaired, e.g., during cell cycle re-entry. Our observations demonstrate that Taz is potently induced during aging of the blood system and most prominently in the LT-HSC compartment where it contributes to age-dependent gene expression changes and functional decline of hematopoietic stem cells.

Citation Format: Anna Mura-Meszaros, Florian Heidel, Bjoern von Eyss. Taz regulates aging of hematopoietic stem cells [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 A10.
Abstract A12: YAP and cancer stem cells in basal-like breast cancer
Authors: Quinn Hazel M. , Vogel Regina , Birchmeier Walter
Published: September 2020

Cancer stem cells (CSCs) are responsible for minimal residual disease tumor relapse. Appearing as the optimal target for therapy, these cells are very difficult to target due to their ability to resist proapoptotic stimuli. In order to overcome this evolutionary stem cell property, elucidation of the mechanistic regulation of these survival pathways is crucial. Previously we identified the CSC population in our model of basal-like breast cancer. Using targeted therapies directed at the tumor-driving pathway (Wnt and Met), we have shown that tumors initially respond to this therapy; however, they quickly become resistant. We have now confirmed that the Hippo transducer, Yap, is active in Wnt-Met tumors. The activity in the CSC population was increased compared to that of bulk tumor cells, indicating that Yap could be an option for anti-CSC therapy. Based on this, we utilized an inhibitor of Yap, verteporfin (VP). Treatment of 3D cultured Wnt-Met cells with VP inhibited CSC survival, showing that Yap is essential for tumor initiation. qPCR analysis of treated, self-renewing spheres showed that Yap and Wnt control distinct sets of stem cell genes. Only combination treatments sufficiently inhibited these genes. We now investigate the relationship between Wnt and Yap to understand how they cooperate and to identify a targetable bottleneck between the two pathways.

Citation Format: Hazel M. Quinn, Regina Vogel, Walter Birchmeier. YAP and cancer stem cells in basal-like breast cancer [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 A12.
Abstract A18: Location, location, location: Avenues to regulating Hippo
Authors: Calses Philamer C. , Pham Victoria , Verschueren Erik , Kljavin Noelyn , Chang Matt , Seidel Kerstin , Haley Benjamin , Pham Trang , Noland Cameron , Hinkle Trent , Hagenbeek Thijs , Lill Jennie , Dey Anwesha
Published: September 2020

The TEAD family of the transcription factors (TEAD1-4) are the major transcription factors for YAP/TAZ transcription activators in the Hippo pathway. TEADs regulate many biologic processes, including development, tissue homeostasis, and tumorigenesis by regulating target genes involved in cellular proliferation and survival. Amplification or upregulation of YAP/TAZ/TEAD correlates with poor prognosis in cancer patients. The mechanisms of TEADs regulation and localization remain largely unknown. TEADs contain a highly conserved nuclear localization signal (NLS) embedded in the DNA binding domain (DBD). Overexpression of an NLS mutant TEAD in a Hippo-dependent cancer cell line is dominant negative and suppresses cellular proliferation and tumor growth. These results suggest that TEAD nuclear expression is regulated by a highly conserved NLS that may be important in the development of cancer. In light of these results, we have identified physiologic conditions where TEADs are expressed in the cytoplasm and the nucleus. By performing comparative immunoprecipitation-mass spectrometry between WT and NLS mutant TEAD, we found novel interacting partners for cytoplasmic and nuclear TEAD, thus uncovering novel roles for TEADs in cancer.

Citation Format: Philamer C. Calses, Victoria Pham, Erik Verschueren, Noelyn Kljavin, Matt Chang, Kerstin Seidel, Benjamin Haley, Trang Pham, Cameron Noland, Trent Hinkle, Thijs Hagenbeek, Jennie Lill, Anwesha Dey. Location, location, location: Avenues to regulating Hippo [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 A18.
Abstract A34: Identification of a MAP kinase that regulates YAP abundance
Authors: Park Sanghyun , Kim Joon
Published: September 2020

Purpose: Recent evidence suggests that YAP and its paralog TAZ play key roles in tumoriogenesis, tumor progression, and chemotherapeutic resistance. Although verteporfin is known to inhibit YAP by suppressing its interaction with the transcription factor TEAD, it has low efficacy and adverse effects including photosensitivity. Therefore, identification of other molecular targets is essential for developing anticancer drugs to treat YAP-driven cancers.

Method: Image-based whole-genome siRNA library screening was performed to identify genes that regulate the abundance of nuclear YAP protein. LATS1/2-null RPE1 cells were used to exclude the influence of cell density that controls the Hippo pathway. Several kinases were identified as positive regulators of nuclear YAP/TAZ intensity. Among them, a member of the MAP kinase family was selected for further study to investigate its YAP-regulating mechanism and its role in BRAF inhibitor-resistant cutaneous melanoma, which has high YAP/TAZ activity. Direct phosphorylation of YAP by the MAP kinase was detected by in vitro kinase assay and LC-MS/MS. Roles of the kinase in BRAF inhibitor-resistant melanoma cells were evaluated by CCK assay, scratch assay, tumor xenograft, and apoptosis analysis.

Results: We found that RNAi-mediated inhibition of the MAP kinase decreases total YAP/TAZ protein levels and their transcriptional activity. The MAP kinase physically interacts with and phosphorylates YAP. This phosphorylation influences ubiquitination and lysosomal degradation of YAP. Knockdown of the MAP kinases decreases proliferation, migration, and tumor formation of BRAF inhibitor-resistant melanoma cells. In addition, inhibition of the kinase reverses BRAF inhibitor resistance.

Conclusion: These results suggest that the MAP kinase is a novel positive regulator of YAP acting independently of the Hippo pathway. We propose that inhibition of the kinase is an efficient way of suppressing YAP/TAZ and the kinase can serve as a therapeutic target for YAP/TAZ-driven cancers.

Citation Format: Sanghyun Park, Joon Kim. Identification of a MAP kinase that regulates YAP abundance [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 A34.
Abstract A40: Hippo signaling in cancer development
Authors: Han Han , Seo Gayoung , Vargas Rebecca , Wang Wenqi
Published: September 2020

The Hippo pathway is known to control cell growth and stimulates cell apoptosis, two key events that are frequently dysregulated in tumorigenesis. Long-term YAP activation results in cell transformation and tumor development, suggesting the power of the Hippo pathway in control of cancer initiation and progression. Genetic mouse models fully support an oncogenic role for YAP and a tumor-suppressive function for the Hippo pathway components. Given the enormous implications of the Hippo pathway in cancer, great efforts have been made in dissecting the genetic mutations of the Hippo pathway components in cancer development and searching for effective approaches to target YAP for therapeutic intervention. Actually, elevated YAP expression and nuclear enrichment have been observed in many types of cancers including liver, breast, lung, ovary, colon, brain, and others. Interestingly, our recent study demonstrated that Hippo signaling deficiency enables the development of an active YAP addiction in cancer cells, which provides an opportunity to develop new targeted strategies for the cancers with deficient Hippo signaling.

Citation Format: Han Han, Gayoung Seo, Rebecca Vargas, Wenqi Wang. Hippo signaling in cancer development [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 A40.
Abstract A43: Identification of novel YAP/TAZ regulators in metastatic cancer
Authors: Xiao Yuxuan , Norton Emily , Warren Janine , Lamar John
Published: September 2020

Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are the downstream effectors of the Hippo tumor inhibition pathway. Studies from us and others show that inappropriate activation of YAP and/or TAZ promotes tumor growth and metastasis. Increased YAP and/or TAZ expression and nuclear localization is observed in many human cancers and often predict poor prognosis. Mutations in Hippo-YAP/TAZ pathway exist but are not common enough to explain the frequently observed increase of YAP/TAZ expression and activity in cancer. Therefore, we hypothesize that other cancer-associated pathways cause aberrant YAP/TAZ activation in cancer cells, and identifying such pathways would provide targets for treatment of YAP/TAZ-driven cancers. Furthermore, transcriptional cofactors are often difficult to target and YAP/TAZ have important functions in adult tissues. Therefore, targeting YAP/TAZ regulators may provide a nonsystemic path of targeting YAP/TAZ that could avoid severe adverse effects. To identify novel YAP/TAZ regulators in metastatic cancer cells, we screened a lentiviral shRNA library that targets 124 cancer-associated genes in A375 MA2 (MA2) cells using a YAP/TAZ-TEAD (TEA domain family members) transcriptional reporter assay. We found that 62 genes are able to regulate YAP/TAZ activity in MA2, 24 of which are already established YAP/TAZ regulators, which supports the effectiveness of our approach. The other 38 genes have not been reported previously, which are potential novel YAP/TAZ regulators. We subsequently validated these candidate YAP/TAZ regulators by examining the expression of the YAP/TAZ target gene CTGF by qPCR and phosphorylation of YAP and TAZ by Western blot in MA2 and other metastatic cell lines. We are currently pursuing several of these novel YAP/TAZ regulators to determine the mechanism through which they regulate YAP/TAZ activity and test their role in metastasis. Our results suggest that YAP/TAZ regulators are cell type and context specific and may also functionally compensate for each other. Therefore, it is critical to identify a “core” group of YAP/TAZ regulators that are important in multiple cancer types. This could provide new strategies to more effectively inhibit YAP/TAZ activation and treat metastatic cancer.

Citation Format: Yuxuan Xiao, Emily Norton, Janine Warren, John Lamar. Identification of novel YAP/TAZ regulators in metastatic cancer [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 A43.
Abstract A45: In silico drug discovery targeting Hippo pathway and YAP-TEAD protein-protein interactions for small-molecule anticancer agent
Authors: Jongwan Kim , Lim Hocheol , No K.T.
Published: September 2020

The Hippo pathway is one of the important pathways regulating tissue growth and proliferation. Dysregulation of this pathway can result in overgrowth of phenotypes because of a malfunction of stem cell proliferation, differentiation, and apoptosis, which are directly involved with components to cancer cell developments. Protein-protein interaction (PPI) in YAP (Yes-associated protein) and TEAD (transcriptional enhancer associate domain) is a key interaction that regulates cancer cell growth in the Hippo pathway. Regulating YAP-TEAD protein-protein interaction by small molecules could play an important role in cancer therapy. In this study, we identified small molecules inhibiting YAP-TEAD interactions by an in silico approach. Molecular hits were identified by pharmacophore-based virtual screening. The hit compounds from virtual screening were tested by surface plasmon resonance (SPR), Luciferase activity test. As a result, several hit compounds were identified to inhibit YAP-TEAD protein-protein interactions. These selected hit compounds were also evaluated by molecular docking study and FMO (fragment molecular orbital) binding energy calculations. In our overall results, we identified hit compounds to inhibit YAP-TEAD PPIs and also analyzed hit compounds’ binding interaction energy to TEAD protein.

Citation Format: Kim Jongwan, Hocheol Lim, K.T. No. In silico drug discovery targeting Hippo pathway and YAP-TEAD protein-protein interactions for small-molecule anticancer agent [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 A45.
Abstract B04: Hepatic cholesterol upregulates TAZ in nonalcoholic steatohepatitis
Authors: Wang Xiaobo , Radhakrishnan Arun , Covey Douglas , Horton Jay , Schwabe Robert , Tontonoz Peter , Guan Kun-Liang , Tabas Ira
Published: September 2020

Nonalcoholic steatohepatitis (NASH) has emerged as the leading cause of chronic liver disease worldwide and is rapidly increasing in prevalence owing to the obesity epidemic. Incomplete understanding of the mechanisms of NASH progression, particularly fibrosis, has limited the design of therapeutic strategies. My previous published study indicated that a transcription factor in hepatocytes called TAZ (WWTR1) contributes to steatosis-to-NASH progression. However, the mechanism that induces high TAZ expression in NASH liver remains unknown. The objective of this study is to understand the mechanism of hepatocyte TAZ upregulation in NASH, which may suggest novel therapeutic strategies to prevent NASH and its comorbidities. We hypothesize that hepatic cholesterol upregulates TAZ through PKA-Ca2+-RhoA mediated Hippo pathway inactivation. In vitro, we use liposome to deplete and replete cholesterol in hepatocyte cells and check downstream signaling pathway that includes cholesterol translocation, Ca2+ signaling, RhoA activity, Hippo pathway, and E3 ligase activity. In vivo, we used different concentration cholesterol diet to build the correlation between increased TAZ/liver fibrosis and cholesterol, and AAV8-H1 mediated hepatic-gene silencing to prove the functions of the molecules in the pathway, including AsterB/C, RhoA, and Trcp. My data suggest that hepatic cholesterol accumulation is associated with liver fibrosis and increased TAZ. In addition, cholesterol loading of hepatocytes increases TAZ, and cholesterol depletion decreases TAZ in a proteasome/Trcp-dependent manner. Mechanistically, Hippo pathway kinase-Lats2 inhibits TAZ expression on the post-transcriptional level through TAZ phosphorylation. Moreover, intracellular cholesterol increases cytosolic Ca2+ and activates RhoA signaling, then represses Lats2 activity, so TAZ degradation is inhibited and total protein level increases. Finally, blocking cholesterol translocation from cell membrane to intracellular suppresses the pathway and inhibits TAZ level. We conclude that hepatic cholesterol induces high-level TAZ in NASH through PKA-Ca2+-RhoA mediated Hippo pathway inactivation, and cholesterol transportation plays an important role during this process. These data provide a plausible mechanism underlying liver cholesterol as a risk factor for NASH fibrosis and suggest new therapeutic interventions to block NASH fibrosis and to prevent NASH-associated HCC.

Citation Format: Xiaobo Wang, Arun Radhakrishnan, Douglas Covey, Jay Horton, Robert Schwabe, Peter Tontonoz, Kun-Liang Guan, Ira Tabas. Hepatic cholesterol upregulates TAZ in nonalcoholic steatohepatitis [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 B04.
Abstract B06: Development of selective LATS1/LATS2 inhibitors for the pharmacologic modulation of the Hippo signaling pathway
Authors: Ceribelli Michele , Morris Patrick , Duveau Damien , Tosto Frances A. , Hoyt Scott , Thomas Craig J.
Published: September 2020

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.
Abstract B09: Selective depletion of YAP1 with next-generation (constrained ethyl-cEt) antisense oligonucleotides results in tumor regression in mouse models of HCC with YAP1 activation
Authors: Kim Youngsoo , Schmidt Joanna , Jo Minji , Yi Jing , Luo Xiaolin , Fleming Rachel , Yim Sun Young , Lee Ju-Seog , Mornia Brett P. , Johnson Randy L. , Mills Gordon , MacLeod A. Robert
Published: September 2020

The Hippo pathway plays important roles in tissue homeostasis. When dysregulated in adult tissues, however, it contributes to the development of a variety of cancers including hepatocellular carcinoma (HCC). YAP1 is the final regulator of the Hippo pathway and is associated with poor prognosis of HCC patients, and more recently has been suggested to be involved in tumor immune evasion. Here, we employed next-generation constrained ethyl (cEt) antisense oligonucleotides (ASOs) to selectively target YAP1 in the genetically engineered mouse (GEM) model of HCC (Salvador KO mice) as well as in a spontaneous carcinogen-induced model of HCC (diethylnitrosamine, DEN-induced HCC). In YAP1-activated Salvador KO mice with established HCC tumors, systemic treatment with YAP1 ASO at 25 mg/kg resulted in a marked reduction in YAP1 protein level in tumor (>90%) and regression of existing tumors, in contrast to the tumors in vehicle or control ASO-treated animals that significantly increased in size. In the DEN-induced HCC model, YAP1 was strongly activated in tumors as demonstrated by nuclear localization of YAP1 protein and the gene signature we previously identified (SOH). YAP1 ASOs produced either complete tumor stasis when DEN tumors were grown in immune-deficient mice, or complete regressions when tumors were grown in immune-competent animals. In accordance with this, YAP1 ASO treatment resulted in significant infiltration of T cells and myeloid lineage cells in the tumors in immune-competent mice. Importantly, when tumor-bearing animals were taken off the treatment after tumor regression was observed with YAP1 ASO, the antitumor response was sustained for more than 1 year, with 4 out of 16 animals remaining tumor-free in YAP1 ASO group. Furthermore, YAP1 ASO in combination with anti-PD1 antibody led to enhanced antitumor activity in the same tumor model. Collectively, these results suggest a potential therapeutic use of YAP1 ASO in the treatment of HCC with YAP1 activation as a single agent or in combination with immuno-oncology drugs. Broad screens have identified the human YAP1 ASO clinical development compound YAP1RX.

Citation Format: Youngsoo Kim, Joanna Schmidt, Minji Jo, Jing Yi, Xiaolin Luo, Rachel Fleming, Sun Young Yim, Ju-Seog Lee, Brett P. Mornia, Randy L. Johnson, Gordon Mills, A. Robert MacLeod. Selective depletion of YAP1 with next-generation (constrained ethyl-cEt) antisense oligonucleotides results in tumor regression in mouse models of HCC with YAP1 activation [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 B09.
Abstract B13: Cancer metabolism sensitizes metformin treatment by targeting the Hippo-YAP/TAZ pathway
Authors: Park Jae Hyung , Park Hyun Woo
Published: September 2020

The biologic significance and deregulation of the Hippo pathway during organ growth and tumorigenesis have received a surge of interest in the past decade. The Hippo pathway core kinases, MST1/2 and LATS1/2, are tumor suppressors that inhibit the oncogenic nuclear function of YAP/TAZ and TEAD. Dysregulation of Hippo pathway leads to tumorigenesis via hyperactivation of YAP/TAZ. In this study, we focused on how differential metabolic stress response (DMSR) between normal and cancer cells, such as the Warburg effect, could synergize with the anticancer effect of metformin to regulate the Hippo pathway. Surprisingly, metformin treatment combined with low glucose concentration activates the Hippo pathway, which induces YAP/TAZ phosphorylation, loss of cell adhesion, and cell death in a cancer cell-specific manner. Using CRISPR KO cells, we show that YAP/TAZ inhibition by metformin in combination with glucose restriction is AMPK and p38 MAPK-independent, but Hippo-dependent. Therefore, these results demonstrate that DMSR-based glucose restriction and metformin cotreatment can be a novel therapeutic approach to treat cancer via inhibition of cancer cell-specific YAP/TAZ activity.

Citation Format: Jae Hyung Park, Hyun Woo Park. Cancer metabolism sensitizes metformin treatment by targeting the Hippo-YAP/TAZ 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 B13.
Abstract B16: Silence of Hippo pathway induces protumoral immunity: New therapeutic target of glioblastomas
Authors: Kim Eui Hyun , Kang Seok-Gu , Lee Ju-Seog
Published: September 2020

The Hippo pathway plays a critical role in various cancers, but its role in glioblastoma (GBM) has not been properly addressed. To assess the clinical relevance of the Hippo pathway in GBM, we generated a core gene expression signature reflecting silence of the Hippo pathway (SOH), and validated it in GBM patient samples from The Cancer Genome Atlas (TCGA) and in a separate cohort, revealing that SOH signature was associated with poor prognosis in GBM in both cohorts. Expression levels of CTGF and CYR61, the most reliable and well-known downstream targets of YAP1 (a target of the Hippo pathway), were markedly increased in GBM with the SOH signature. GBM showing the SOH signature was associated with an increased Immune signature score (ISS), suggesting that these patients might be good potential candidates for immunotherapy. Genes differentially expressed between samples with the SOH signature and samples with an active Hippo signature showed that many inhibitory immune checkpoint genes, such as CTLA4, PD-1, and PD-L2, were upregulated in the SOH subgroup, suggesting that YAP1/TAZ may induce resistance of cancer cells to host immune response in GBM. Also, many M2-polarized microglial markers and innate immune pathways were activated in the SOH subgroup. The SOH signature was strongly correlated with the TGF-β signature, which was also associated with a high ISS and mesenchymal features. In summary, SOH induces protumoral immunity in various ways, which may be new immunotherapeutic targets of GBM.

Note: This abstract was not presented at the conference.

Citation Format: Eui Hyun Kim, Seok-Gu Kang, Ju-Seog Lee. Silence of Hippo pathway induces protumoral immunity: New therapeutic target of glioblastomas [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 B16.
Abstract B29: AXL Inhibitor TP-0903 attenuates TGFβ-Hippo signaling in lung adenocarcinoma cells
Authors: Taverna Josephine A. , Hung Chia-Nung , Lin Chun-Lin , Osmulski Pawel A. , Gaczynska Maria E. , Wang Chiou-Miin , Lucio Nicholas D. , Chen Meizhen , Chou Chih-Wei , Nazarullah Alia , Lampkin Shellye R. , Qiu Lianquin , Bearss David J. , Warner Steve , Mouritsen Lars , Wade Mark , DeArmond Daniel , Mesa Ruben , Kirma Nameer , Huang Tim H.-M.
Published: September 2020

Background: Non-small cell lung cancer (NSCLC) is a molecularly heterogeneous disease with a high propensity for drug resistance and metastasis. AXL, a member of the Tyro3-AXL-Mer (TAM) family of receptor tyrosine kinases, is a central regulator of epithelial-to-mesenchymal transition (EMT) and enables tumor cells to invade and acquire drug resistance. AXL is overexpressed in NSCLC and its expression correlates positively with tumor invasion, drug resistance, and negatively predicts overall survival. We mechanistically interrogated the effects of the AXL inhibitor, TP-0903, on EMT in NSCLC cells using transcriptomic and proteomic profiling.

Methods: Atomic force microscopy, Western blot analysis, RNA sequencing, and mass cytometry (CyTOF) were used to evaluate the phenotypic, transcriptomic, and proteomic profiles of A549 cells treated with 40 nM TP-0903 or shAXL knockdown. A549 and H1650 NSCLC xenograft models were used to explore the consequences of AXL inhibition in vivo.

Results: As expected, TP-0903 treatment attenuated AXL signaling and downstream phosphorylation in the A549 cells. Interestingly, the treatment also reduced gene expression responses to TGFβ-Hippo signaling by disrupting the transcriptional complexes formed by SMAD2/3, SMAD4, YAP1, and TAZ. Consistent with AXL inhibition, TP-0903 reversed the mesenchymal phenotype in A549 and H2009 cell lines and decreased their migratory potential in culture. The CyTOF analysis on TP-0903-treated cells identified resistant clones overexpressing TGFβ receptor II (TGFBR2) and TAZ proteins and displaying hybrid EMT phenotypes. TP-0903 was also active in suppressing A549 or H1650 tumor growth in vivo.

Conclusions: We are the first to report the interplay between AXL and TGFβ-Hippo signaling axis. TP-0903 has excellent therapeutic promise in NSCLC and we speculate that TP-0903 can target mesenchymal transitional states in NSCLC, possibly through the inhibition of the AXL-TGFβ-Hippo signaling axis.

Citation Format: Josephine A. Taverna, Chia-Nung Hung, Chun-Lin Lin, Pawel A. Osmulski, Maria E. Gaczynska, Chiou-Miin Wang, Nicholas D. Lucio, Meizhen Chen, Chih-Wei Chou, Alia Nazarullah, Shellye R. Lampkin, Lianquin Qiu, David J. Bearss, Steve Warner, Lars Mouritsen, Mark Wade, Daniel DeArmond, Ruben Mesa, Nameer Kirma, Tim H.-M. Huang. AXL Inhibitor TP-0903 attenuates TGFβ-Hippo signaling in lung adenocarcinoma cells [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 B29.
Abstract B41: Role of AIB1 in YAP-TEAD signaling in the progression of early-stage breast cancer
Authors: Kushner Max Harley , Graham Garrett , Sharif Ghada , Kietzman Will , Glasgow Eric , Sangupta Surojeet , Wellstein Anton , Riegel Anna Tate
Published: September 2020

AIB1 is an oncogenic transcription coactivator that is overexpressed and amplified in breast cancer and is known to interact with TEAD. AIB1, like YAP, is critical to the development and progression of triple-negative breast cancer, yet it is unclear to what extent the AIB1-TEAD interaction promotes the progression of triple-negative breast cancer. Herein, we confirm by ChIP and ChIP-seq that AIB1 is recruited to the chromatin with TEAD in complex with the YAP oncogene. In doing so, AIB1 potentiates YAP activity on a subset of TEAD target genes known to be critical to driving epithelial-to-mesenchymal and invasive behaviors in early-stage and advanced triple-negative breast cancer. Loss of AIB1 reverts the oncogenic phenotype conferred by YAP and attenuates 3D matrix invasion, migration, and endothelial extravasation, suggesting AIB1 may act as an oncogene in triple-negative breast cancer through YAP-TEAD. We find AIB1-YAP coregulation of a subset of oncogenic target genes that is determined by relative enrichment and global proximity of Oct1/POU2F1 binding motifs within target promoters. Thus, AIB1 is critical to maintaining YAP driven EMT and invasive phenotypes during the development and progression of triple-negative breast cancer.

Citation Format: Max Harley Kushner, Garrett Graham, Ghada Sharif, Will Kietzman, Eric Glasgow, Surojeet Sangupta, Anton Wellstein, Anna Tate Riegel. Role of AIB1 in YAP-TEAD signaling in the progression of early-stage breast cancer [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 B41.
Abstract B42: YAP1/Hippo pathway and SWI/SNF as critical players in squamous cancers and normal development
Authors: Ellisen Leif , Tata Purushothama Rao , Zhao Rui , Rajagopal Jayaraj , Saladi Srinivas Vinod
Published: September 2020

Cancer development is a multistep process involving deregulation of transcription/epigenetic factors and signaling pathways. Accordingly, gene expression lies at the nexus of oncogenic cellular processes. The recent advent of The Cancer Genome Atlas (TCGA) has shed light on multiple genes involved in signaling, transcription, and other cellular processes that are subjected to genomic alterations in multiple cancers. These oncogenic drivers are involved in regenerative proliferation, metastasis, and other cellular processes that contribute to tumorigenesis. Epithelial-stromal interactions and the infiltrating immune response contribute significantly to tumor progression. Our recent studies revealed Hippo pathway activation as a critical component in ACTL6A (subunit of SWI/SNF chromatin remodeling complex) amplified squamous cancers. Our published genetic studies indicate that YAP is a key mediator of the oncogenic function of ACTL6A/p63 in head and neck squamous cancers. P63 is a master transcription factor in epithelial development. We have shown that YAP and p63 biochemically interact to maintain airway architecture and regeneration. YAP and p63 deletion are phenocopies and are required to maintain normal respiratory epithelial stem cells. P63/ACTL6A act as upstream regulators of YAP in squamous cancers. ACTL6A and p63 physically interact and cooperatively control a transcriptional program that promotes proliferation and suppresses differentiation, through activation of the Hippo-YAP pathway via regulators including WWC1 and GPRC5A. Consequently, loss of ACTL6A or p63 in tumor cells induces YAP phosphorylation and inactivation, associated with growth arrest and terminal differentiation, all phenocopied by WWC1 overexpression. YAP activation predicts poor patient survival in squamous cancers. These findings are also in keeping with the emerging role of the YAP pathway as a tumor driver in multiple contexts. Given interest in YAP as a therapeutic target, our studies reveal contribution of p63 and TEAD to YAP function as evident from global transcription profiling. Our most recent studies also indicate contribution of YAP in tumor progression by evading host immune response. All these indicate an important intrinsic role of YAP in development and disease, making it one of the ideal therapeutic targets to control cancer.

Citation Format: Leif Ellisen, Purushothama Rao Tata, Rui Zhao, Jayaraj Rajagopal, Srinivas Vinod Saladi. YAP1/Hippo pathway and SWI/SNF as critical players in squamous cancers and normal development [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 B42.
Abstract B43: Division of labor between YAP and TAZ in lung cancer
Authors: Shreberk-Shaked Michal , Dassa Bareket , Di Agostino Silvia , Nuriel Yarden , Wigoda Noa , Rotkopf Ron , Blandino Giovanni , Aylon Yael , Oren Moshe
Published: September 2020

Paralogs originating from a gene duplication event can diverge and accumulate changes that contribute to a division of labor. Therefore, we wished to explore differential activities of YAP and TAZ. Traditionally, research has tended to consider YAP and TAZ as functionally redundant transcriptional cofactors, with similar biologic impact. However, there is growing evidence that each of them possesses distinct attributes. Hence, it is of great interest to find out whether YAP and TAZ have nonredundant involvement in cancer. Lung cancer is the leading cause of cancer-related deaths worldwide. Despite significant progress, the 5-year survival is still lower than 15%. Therefore, there is an urgent need for novel lung cancer biomarkers. Recent evidence suggests that YAP/TAZ play important roles in lung tumorigenesis. We explored differences in the transcriptional programs regulated by YAP and TAZ in lung adenocarcinoma cells, and the physiologic implications of such differences. Towards this aim, we performed RNA-sequencing in lung cancer cells following knockdown of either YAP or TAZ. Our analysis revealed a broad spectrum of expression patterns, including genes that are regulated preferentially by either YAP, TAZ, or both. Interestingly, a group of genes related to extracellular matrix remodeling was substantially regulated by TAZ but not YAP, while cell cycle- and proliferation-related genes were affected mainly by YAP but not TAZ. These differences in gene regulation correlated with functional differences between YAP and TAZ. Knockdown of TAZ strongly affected cell migration, whereas depletion of YAP had only mild effects on migration. Conversely, YAP silencing resulted in reduced proliferation compared to TAZ silencing. Importantly, YAP- and TAZ-dependent transcriptomes were correlated to lung cancer patient survival. In summary, our findings suggest that YAP and TAZ have distinct but complementary roles, which are associated with different cancer features. Hopefully, the identification of different activities and downstream effects of YAP and TAZ may enable more refined stratification of lung cancers and provide clues for the development of more effective targeted therapies.

Citation Format: Michal Shreberk-Shaked, Bareket Dassa, Silvia Di Agostino, Yarden Nuriel, Noa Wigoda, Ron Rotkopf, Giovanni Blandino, Yael Aylon, Moshe Oren. Division of labor between YAP and TAZ in lung cancer [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 B43.
Abstract IA01: Targeting developmental pathways in cancer
Authors: de Sauvage Frederic
Published: September 2020

Signaling pathways such as Notch, Wnt, Hedgehog, and Hippo play critical roles during embryonic development through modulation of proliferation, cell migration, and differentiation. In adults, these pathways remain important in regulating stem cell function during normal tissue homeostasis. However, inappropriate activation either through mutation or misexpression can result in tumorigenesis, making potential targets for the development of novel targeted therapeutics. The Hedgehog pathway provides a textbook example of this scenario where aberrant activation of the pathway through Patched (PTCH) loss of function mutations or Smoothened (SMO) activating mutation leads to the development of various types of cancers such as basal cell carcinoma or medulloblastoma. These observations drove the development of Hedgehog pathway inhibitors (HPI) for the treatment of various cancers, resulting in the approval of two SMO inhibitors, vismodegib and sonidegib, for the treatment of advanced BCC. Lessons learned from targeting the Hedgehog pathway may inform the development of future therapies.

Citation Format: Frederic de Sauvage. Targeting developmental pathways in cancer [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 IA01.
Abstract IA08: Regulation of YAP by tyrosine phosphorylation of core Hippo pathway components: Lessons learned from the GNAQ oncogene and FAK
Authors: Gutkind J. Silvio
Published: September 2020

Dysregulation of the Hippo signaling pathway and the consequent YAP1 activation is a frequent event in human malignancies, yet the underlying molecular mechanisms are still poorly understood. In prior studies we have focused on uveal melanoma, the most frequent eye malignancy in adults that is caused by constitutively active mutations in the GNAQ oncogenes, which encode Gαq and Gα11 heterotrimeric G protein α subunits. We found that GNAQ promotes the activation of YAP1 by a mechanism involving the activation of RhoA by the direct association of Gαq to TRIO, a Rho-GEF, and the consequent stimulation of acting polymerization and the release of YAP from its complex with AMOT. We also showed that YAP1 is essential for uveal melanoma cell growth, thus representing a potential therapeutic target for this malignancy. However, no effective and safe YAP1 inhibitors are currently available. Using a novel integrated bioinformatics pipeline, we recently found that PTK2, encoding Focal Adhesion Kinase (FAK), represents a candidate synthetic lethal gene with GNAQ activation (Feng et al., Cancer Cell 2019). We found that Gαq activates FAK through the TRIO-RhoA noncanonical Gαq-signaling, and that genetic ablation or pharmacologic inhibition of FAK inhibits uveal melanoma growth. Surprisingly, analysis of the FAK-regulated transcriptome indicated that GNAQ stimulates YAP1 through FAK. Dissection of the underlying mechanism revealed that FAK regulates YAP1 by tyrosine phosphorylation of MOB1, thereby disrupting MOB1/LATS association and core Hippo pathway signaling. By coupling the power of an unbiased computational pipeline to the unique genetic landscape of uveal melanoma, we uncovered a molecular framework regulating YAP1 and identified FAK as a druggable signaling hub downstream from GNAQ in uveal melanoma. Our findings established FAK as a potential therapeutic target for uveal melanoma and other diseases that may involve FAK activation and unrestrained YAP1 function. Further studies on how multiple growth promoting and oncogenic tyrosine kinase signaling networks may converge to phosphorylate core Hippo pathway components to activate YAP1 in cancer will be also discussed.

Citation Format: J. Silvio Gutkind. Regulation of YAP by tyrosine phosphorylation of core Hippo pathway components: Lessons learned from the GNAQ oncogene and FAK [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 IA08.
Abstract IA09: The crosstalk between Hippo-YAP/TAZ and PTEN-AKT signaling in liver metabolic dysregulation and tumorigenesis
Authors: Jeong Sun-Hye , Lim Dae-Sik
Published: September 2020

Several in vitro studies have suggested that Hippo-YAP/TAZ signaling regulates the AKT pathway components PI3K and PTEN. Increased YAP expression in human liver tumors is associated with high levels of phosphorylated AKT. This suggests that crosstalk between the Hippo and AKT pathways may be important in the maintenance of functional liver homeostasis. The nature of such crosstalk has thus far remained unclear. Here, we investigated the possible role of crosstalk between Hippo-YAP/TAZ and PTEN-AKT pathways in the liver. Deletion of both Pten and Sav1 in the liver (DKO) accelerates the development of nonalcoholic fatty liver disease (NAFLD) and liver cancer in mice through excessive activation of AKT. At the molecular level, activation of YAP and TAZ amplifies AKT signaling through direct upregulation of IRS2 expression. Deletion of YAP/TAZ or activation of Hippo attenuated development of fatty liver in these DKO mice by downregulating IRS2. Notably, increased YAP/TAZ expression was also associated with high level of IRS2-pAKT in the patient. Moreover, treatment with the AKT inhibitor MK-2206 attenuated NAFLD development and tumorigenesis in DKO mice. Our findings suggest that crosstalk between Hippo and AKT pathways at the level of YAP/TAZ-IRS2 contributes to the development and progression of NAFLD to liver cancer, and they therefore provide a basis for the development of new therapeutic interventions.

Citation Format: Sun-Hye Jeong, Dae-Sik Lim. The crosstalk between Hippo-YAP/TAZ and PTEN-AKT signaling in liver metabolic dysregulation and tumorigenesis [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 IA09.