YAP Target Genes Research Articles

PRMT1-mediated arginine methylation promotes YAP activation and hepatocellular carcinoma proliferation.

The activity of Hippo signaling is commonly dysregulated in various human malignancies, including hepatocellular carcinoma (HCC). YAP, the key effector of Hippo pathway, is regulated through several posttranslational modifications. However, the mechanism by which YAP is regulated by arginine methylation remains unknown. In this study, immunoprecipitation and mass spectrometry were used to identify the arginine methylation site of YAP in HCC cells. The transcriptional activity of YAP and TEAD were further characterized by real-time qPCR and immunofluorescence assay, and a subcutaneous and orthotopic tumor mouse model was used to assess the effect of PRMT1-knockdown on HCC tumor growth. The result of mass spectrometry analysis identified that YAP was methylated at arginine 124. Moreover, we found that arginine methyltransferase PRMT1 interacted with YAP to mediate its arginine methylation, thus inhibited YAP phosphorylation and promoted YAP activity in the nucleus. PRMT1 was up-regulated in HCC tissues and positively associated with the expressions of YAP target genes. Silencing PRMT1 in HCC cells inhibited cell proliferation and tumor growth, while PRMT1-overexpression promoted HCC growth through YAP methylation. Our study reveals that PRMT1-mediated arginine methylation at R124 is mutually exclusive with YAP S127 phosphorylation, thereby facilitating YAP activity in the nucleus and promoting tumorigenesis in HCC.

CD47, a novel yap target gene, contributes to hepatic stellate cell activation and liver fibrosis induced by high-fat diet

CD47, a novel yap target gene, contributes to hepatic stellate cell activation and liver fibrosis induced by high-fat diet

Exogenous S1P via S1P receptor 2 induces CTGF expression through Src-RhoA-ROCK-YAP pathway in hepatic stellate cells.

Hepatic fibrosis, a prevalent chronic liver condition, involves excessive extracellular matrix production associated with aberrant wound healing. Hepatic stellate cells (HSCs) play a pivotal role in liver fibrosis, activated by inflammatory factors such as sphingosine 1-phosphate (S1P). Despite S1P's involvement in fibrosis, its specific role and downstream pathway in HSCs remain controversial. In this study, we investigated the regulatory role of S1P/S1P receptor (S1PR) in Hippo-YAP activation in both LX-2 cell lines and primary HSCs. Real-time PCR, western blot, pharmacological inhibitors, siRNAs, and Rho activity assays were adopted to address the molecular mechanisms of S1P mediated YAP activation. Serum and exogenous S1P significantly increased the expression of YAP target genes in HSCs. Pharmacologic inhibitors and siRNA-mediated knockdowns of S1P receptors showed S1P receptor 2 (S1PR2) as the primary mediator for S1P-induced CTGF expression in HSCs. Results using siRNA-mediated knockdown, Verteporfin, and Phospho-Tag immunoblots showed that S1P-S1PR2 signaling effectively suppressed the Hippo kinases cascade, thereby activating YAP. Furthermore, S1P increased RhoA activities in cells and ROCK inhibitors effectively blocked CTGF induction. Cytoskeletal-perturbing reagents were shown to greatly modulate CTGF induction, suggesting the important role of actin cytoskeleton in S1P-induced YAP activation. Exogeneous S1P treatment was enough to increase the expression of COL1A1 and α-SMA, that were blocked by YAP specific inhibitor. Our data demonstrate that S1P/S1PR2-Src-RhoA-ROCK axis leads to Hippo-YAP activation, resulting in the up-regulation of CTGF, COL1A1 and α-SMA expression in HSCs. Therefore, S1PR2 may represent a potential therapeutic target for hepatic fibrosis.

Aggressive high-grade NF2 mutant meningiomas downregulate oncogenic YAP signaling via the upregulation of VGLL4 and FAT3/4.

Meningiomas are the most common primary central nervous system tumors in adults. Although generally benign, a subset is of higher grade and ultimately fatal. Around half of all meningiomas harbor inactivating mutations in NF2, leading to deregulation of oncogenic YAP1 activity. While benign NF2 mutant meningiomas exhibit few genetic events in addition to NF2 inactivation, aggressive high-grade NF2 mutant meningiomas frequently harbor a highly aberrant genome. It is unclear if NF2 mutant meningiomas of different grades are equally reliant on YAP activity. We analyzed bulk and single-cell RNA-Seq data from a large cohort of human meningiomas for the expression of YAP1 target genes and Hippo effectors as well as in vitro cell line experiments. Aggressive NF2 mutant meningiomas harbor decreased expression levels of YAP1 target genes and increased expression levels of the YAP1 antagonist VGLL4 and the upstream regulators FAT3/4 compared to their benign counterparts. Decreased expression of YAP1 target genes as well as high expression of VGLL4 and FAT3/4 is significantly associated with an increased risk of recurrence. In vitro, overexpression of VGLL4 resulted in the downregulation of YAP activity in benign NF2 mutant meningioma cells, confirming the direct link between VGLL4 expression and decreased levels of YAP activity observed in aggressive NF2 mutant meningiomas. Our results shed new insight into the biology of benign and aggressive NF2 mutant meningiomas and may have important implications for the efficacy of therapies targeting oncogenic YAP1 activity in NF2 mutant meningiomas.

YAP/TAZ enhances P-body formation to promote tumorigenesis

The role of processing bodies (P-bodies) in tumorigenesis and tumor progression is not well understood. Here, we showed that the oncogenes YAP/TAZ promote P-body formation in a series of cancer cell lines. Mechanistically, both transcriptional activation of the P-body-related genes SAMD4A, AJUBA, and WTIP and transcriptional suppression of the tumor suppressor gene PNRC1 are involved in enhancing the effects of YAP/TAZ on P-body formation in colorectal cancer (CRC) cells. By reexpression of PNRC1 or knockdown of P-body core genes (DDX6, DCP1A, and LSM14A), we determined that disruption of P-bodies attenuates cell proliferation, cell migration, and tumor growth induced by overexpression of YAP5SA in CRC. Analysis of a pancancer CRISPR screen database (DepMap) revealed co-dependencies between YAP/TEAD and the P-body core genes and correlations between the mRNA levels of SAMD4A, AJUBA, WTIP, PNRC1, and YAP target genes. Our study suggests that the P-body is a new downstream effector of YAP/TAZ, which implies that reexpression of PNRC1 or disruption of P-bodies is a potential therapeutic strategy for tumors with active YAP.

YAP/TAZ enhances P-body formation to promote tumorigenesis.

The role of processing bodies (P-bodies) in tumorigenesis and tumor progression is not well understood. Here, we showed that the oncogenes YAP/TAZ promote P-body formation in a series of cancer cell lines. Mechanistically, both transcriptional activation of the P-body-related genes SAMD4A, AJUBA, and WTIP and transcriptional suppression of the tumor suppressor gene PNRC1 are involved in enhancing the effects of YAP/TAZ on P-body formation in colorectal cancer (CRC) cells. By reexpression of PNRC1 or knockdown of P-body core genes (DDX6, DCP1A, and LSM14A), we determined that disruption of P-bodies attenuates cell proliferation, cell migration, and tumor growth induced by overexpression of YAP5SA in CRC. Analysis of a pancancer CRISPR screen database (DepMap) revealed co-dependencies between YAP/TEAD and the P-body core genes and correlations between the mRNA levels of SAMD4A, AJUBA, WTIP, PNRC1, and YAP target genes. Our study suggests that the P-body is a new downstream effector of YAP/TAZ, which implies that reexpression of PNRC1 or disruption of P-bodies is a potential therapeutic strategy for tumors with active YAP.

Aggressive high-grade NF2 mutant meningiomas downregulate oncogenic YAP signaling via the upregulation of VGLL4 and FAT3/4.

Meningiomas are the most common primary brain tumors in adults. Although generally benign, a subset of meningiomas is of higher grade, shows aggressive growth behavior and recurs even after multiple surgeries. Around half of all meningiomas harbor inactivating mutations in NF2. While benign low-grade NF2 mutant meningiomas exhibit few genetic events in addition to NF2 inactivation, aggressive high-grade NF2 mutant meningiomas frequently harbor a highly aberrant genome. We and others have previously shown that NF2 inactivation leads to YAP1 activation and that YAP1 acts as the pivotal oncogenic driver in benign NF2 mutant meningiomas. Using bulk and single-cell RNA-Seq data from a large cohort of human meningiomas, we show that aggressive NF2 mutant meningiomas harbor decreased levels YAP1 activity compared to their benign counterparts. Decreased expression levels of YAP target genes are significantly associated with an increased risk of recurrence. We then identify the increased expression of the YAP1 competitor VGLL4 as well as the YAP1 upstream regulators FAT3/4 as a potential mechanism for the downregulation of YAP activity in aggressive NF2 mutant meningiomas. High expression of these genes is significantly associated with an increased risk of recurrence. In vitro, overexpression of VGLL4 resulted in the downregulation of YAP activity in benign NF2 mutant meningioma cells, confirming the direct link between VGLL4 expression and decreased levels of YAP activity observed in aggressive NF2 mutant meningiomas. Our results shed new insight on the biology of benign and aggressive NF2 mutant meningiomas and may have important implications for the efficacy of therapies targeting oncogenic YAP1 activity in NF2 mutant meningiomas.

Establishment of Anoxia/cytokine-induced Injury and Regeneration in Colonoids

Intestinal epithelial cells (IECs) play key roles in nutrient absorption and fluid secretion, providing a barrier to protect cells. IECs, derived from intestinal stem cells (ISCs), are crucial in maintaining intestinal homeostasis and regeneration, which is crucial in gastrointestinal diseases such as inflammatory bowel disease (IBD). To study epithelial injury/regeneration, primary cultured colonoids were utilized as a model system to mimic injury. They retain the composition and differentiation of the tissue, making it a suitable model for studying homeostasis and regeneration of ISCs in vitro. Anoxia and proinflammatory cytokines frequently coexist and exacerbate one another during intestinal injury. However, anoxic conditions have been mainly ignored in previous research. Thus, we developed a regeneration model using cytokines/anoxia injury to better recapitulate intestinal inflammation. Colonoids were treated with cytokines TNF-α and IFN-γ (5 ng/μL) in anoxic conditions for 24 hours to provoke an injury. After 24 hours of injury, the colonoids were exposed to normal oxygen and media conditions for regeneration. Regeneration was observed for 24, 48, and 72 hours and documented using photomicrographs. Morphological analysis detailed that injury was most severe 24 hours post-injury with the combined induction of cytokines and anoxia resulting in decreased colonoid area and number of buds compared to control groups. RT-PCR analysis detailed mRNA expression of stem cell markers ( Lgr5, Bmi1, and Hopx), Wnt ( Axin2, Myc2, and Ascl2), and YAP target genes ( Sca1 and Ctgf) were increased at peak injury and returned to pre-injury levels at 72 hours of recovery. Regeneration of the colonoids is consistent with in vivo observations, thus confirming the validity of the model. This versatile model can be used to study responses to injury and regeneration of intestinal epithelium and the function of ISCs with applications in intestinal disease therapeutics. University of Maryland College of Agriculture and Natural Resources and the Maryland Agricultural Experiment Station Undergraduate Research Grant Grant #4307572 from the Maryland Stem Cell Research Fund. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Design, Synthesis, and Bioevaluation of Transcriptional Enhanced Assocciated Domain (TEAD) PROTAC Degraders.

Dysregulation of the Hippo pathway has been observed in various cancers. The transcription factor TEAD, together with its coactivators YAP/TAZ, plays a crucial role in regulating the transcriptional output of the Hippo pathway. Recently, extensive research has focused on small molecule inhibitors targeting TEAD, but studies on TEAD degraders are comparatively rare. In this study, we designed and synthesized a series of TEAD PROTACs by connecting a pan-TEAD inhibitor with the CRBN ligand thalidomide. A representative compound, 27, exhibited potent antiproliferative activity against NF2-deficient NCI-H226 cells. It dose-dependently induced TEAD degradation dependent on CRBN and proteasome system and decreased key YAP target genes CYR61 and CTGF expressions in NCI-H226 cells. Further degradation selectivity studies revealed that 27 exhibited more potent activity against TEAD2 compared to those of the other three family members in Flag-TEADs transfected 293T cells. Therefore, 27 may serve as a valuable tool for advancing biological studies related to TEAD2.

USP12 facilitates gastric cancer progression via stabilizing YAP

The dysregulation of Hippo signaling is a crucial factor driving the progression of gastric cancer, making the targeting of the Hippo pathway a promising therapeutic strategy. However, effective drugs targeting the Hippo/YAP axis remain unavailable. Thus, identifying potential therapeutic targets and mechanisms that inhibit the activity of the Hippo/YAP axis in gastric cancer is of paramount importance. The ubiquitination modification of the Hippo/YAP pathway plays a significant role in signaling transduction and cancer progression. In an effort to shed light on effective therapeutic targets, we conducted a screening using a deubiquitinase small interfering RNA library, leading to the identification of USP12 as an important deubiquitinase in the context of Hippo/YAP axis and the progression of gastric cancer. Our bioinformatic analysis further demonstrated a correlation between USP12 and poor survival, as well as a positive association with classical YAP target genes in gastric cancer samples. Notably, USP12 depletion was found to inhibit gastric cancer progression via the Hippo/YAP axis, whereas USP12 overexpression exhibited the opposite effect, promoting gastric cancer growth and enhancing YAP activity. Further studies through immuno-staining and immuno-precipitation assays indicated the nuclear localization of USP12 and its association with YAP to enhance YAP stability. Specifically, our findings revealed that USP12 could inhibit K48-linked poly-ubiquitination of YAP, predominantly at the K315 site. As a result, we have identified a novel regulatory mechanism involving USP12 and Hippo signaling in the progression of gastric cancer, with the potential for blockade of USP12 to materialize as a promising strategy for combating gastric cancer.

Abstract 4266: Evaluation of integrin alpha v beta 5 as a novel target for non-small cell lung cancer via tumor intrinsic and host immune regulation

Abstract Lung cancer is the leading cause of cancer related deaths worldwide, with the most prevalent subtype being non-small cell lung cancer (NSCLC). As only a subset of patients exhibits durable clinical responses to current treatments, including targeted therapy and immunotherapy, new treatment options are needed. Integrins (ITGs) are a diverse class of heterodimeric, transmembrane receptors that bind extracellularly to the extracellular matrix (ECM) and intracellularly to the cytoskeleton to integrate signals bi-directionally between the extracellular environment and cell interior. One of 18 α subunits is paired with one of 6 β subunits to create the 24 known αβ ITGs in mammals. ITGs are expressed at low levels in normal tissue and can be upregulated in cancer cells in response to stress signals in the tumor microenvironment (TME). Altered expression of ITGs is associated with enhanced cell proliferation, metastasis, and immune evasion. Targeting of the diverse ITG family members by inhibitors not specific to individual isoforms has obscured understanding of potentially distinct functions of each heterodimer. Through data mining of a genome wide CRISPR screening publicly available at the DepMap portal, we have identified a novel cancer dependency to ITG αvβ5 in approximately 40% of NSCLC cell lines. Using small molecule ITG αv inhibitors, we have mirrored sensitivity of NSCLC cell lines to their ITG αvβ5 CRISPR scores, suggesting that these inhibitors function specifically through ITG αvβ5 in cell lines. RNA sequencing analysis following αvβ5 inhibition in human NSCLC cell lines revealed downregulation of YAP and E2F1 target genes with alterations in gene ontologies associated with cell cycling and cell death in sensitive, but not resistant, lines. This is consistent with the known function of YAP driving gene transcription promoting cell proliferation and survival, and of E2F1 being implicated in regulating the proliferation and self-renewal of cancer stem cell (CSC). Additional changes to immune processes, specifically perturbations to interferon (IFN) pathways, were identified in response to ITG αvβ5 inhibition, suggesting that ITG αvβ5 may play a role in host anti-tumor immune responses. Pharmacological inhibition of ITG αvβ5 also led to a reduction of tumor growth in NSCLC murine models accompanied with increased CD8 T cell activation. These findings support ITG αvβ5 as a unique target of cancer dependency in NSCLC whose inhibition promotes anti-tumor responses through both tumor intrinsic and host immune regulatory mechanisms. Citation Format: Bitta P. Kahangi, William P. Crosson, Ji-Ann Lee, Camelia Dumitras, Tianhao Zhang, Ramin Salehi-Rad, Linh M. Tran, Michael Palazzolo, Dylan Conklin, Steven M. Dubinett, Bin Liu. Evaluation of integrin alpha v beta 5 as a novel target for non-small cell lung cancer via tumor intrinsic and host immune regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4266.

Abstract 4585: Discovery of ETS-006, a highly potent YAP/TEADs PPI inhibitor with broad anti-tumor activity as a single agent

Abstract The transcription co-activators YAP, pairing with the TEAD family of transcription factors, serve as essential effectors of the conserved Hippo signaling pathway. Hippo kinase cascade controls YAP nuclear entry and binding with TEADs by modulating its phosphorylation and stability. Overexpression and activation of YAP/TAZ are frequently observed in a variety of cancers. It has been well established that YAP aberration contributes to cancer development, metastasis, immunosurveillance escape and drug resistance. Disruption of YAP/TEADs PPI is an effective approach to inhibit YAP transcriptional activity. We previously have reported the identification of YAP/TEADs PPI hit compound through the fragment-based screen and advanced the hit to lead compound ETS-003. Here we reported the preclinical candidate of YAP/TEADs PPI inhibitor ETS-006 with much more potent anti-tumor activity and favorable safety profile. Compared to TEAD palmitoylation inhibitor, ETS-006 could completely disrupt the interaction of YAP/TEADs, resulting in a more profound downregulation of YAP target gene expression. ETS-006 demonstrated more robust anti-tumor activity than the competitor’s YAP/TEADs PPI inhibitor in MPM CDX model with favorable ADME properties. Notably, ETS-006 showed promising efficacy across multiple solid tumor types, including head and neck squamous cell carcinoma, osteosarcoma, and triple-negative breast cancer with urgent unmet clinical needs. In conclusion, ETS-006 is a highly potent and orally available YAP/TEADs PPI inhibitor with broad anti-tumor activity as a single agent. Citation Format: Jiajun Lu, Ming Gao, Wenpei Du, Xiaofei Fan, Jinping Li, Mengya Wang, Lijian Feng, Yingjie Li, Jiaqi Yao, Jiting Lu, Wenqi Cui, Qiangang Zheng, Jidong Zhu. Discovery of ETS-006, a highly potent YAP/TEADs PPI inhibitor with broad anti-tumor activity as a single agent [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4585.

Abstract 1707: YAP-driven epigenetic reprogramming in head and neck cancer

Abstract The ability to measure tumor evolution is difficult due to a lack of model systems able to capture tumor initiation, development, and invasion. We have developed a Dox-inducible spatiotemporally controlled HPV- and YAP-driven model of head and neck squamous cell carcinoma (HNSCC) with a well-defined cancer stem cell population. In order to better understand the epigenetic reprogramming associated with tumorigenesis we have systematically characterized the chromatin accessibility, histone landscape and YAP binding sites in this model. Cell lines were generated from tongue epithelia of normal and Dox-induced tumor-containing mice. Gene expression was measured using RNA-seq and chromatin accessibility was measured using ATAC-seq. CUT&Tag for H3K27me3, H3K27ac, and YAP was used to measure epigenetic reprogramming and YAP-DNA interactions. Differential gene expression shows that genes involved in hormone signaling, glutathione-related metabolism, fatty acid metabolism and p53 signaling were highly enriched in the normal tongue suggesting a change in metabolism upon tumorigenesis.Differential ATAC-seq peaks with higher binding levels in the normal tongue showed enrichment for p53 motifs as well as homebox motifs including Otx2, GSC and CRX suggesting p53 signaling is lost upon carcinogenesis and lineage determination is altered. Motifs for TEAD, AP-1, and KLF5 were enriched in the tumor-specific ATAC-seq peaks showing that YAP is involved in chromatin accessibility changes. CUT&Tag for YAP shows clear YAP binding near canonical YAP target genes including Axl, Ccn1, Ccn2 and Birc3. YAP peaks with higher binding in the normal tongue cell line were enriched for RUNX2 and p63 motifs and peaks higher in the tumor contained KLF5 motifs showing YAP is directly involved in the reprogramming of lineage determination. Multi-omics integration shows YAP-driven chromatin remodeling is associated with differential expression of genes involved in keratin production, EGFR and ER signaling. Direct epigenetic characterization of YAP involvement in tumor initiation is key to understanding the mechanisms of tumorigenesis in head and neck cancer. This work shows that YAP directly binds and affects the expression of genes involved in lineage determination and growth factor signaling to induce cancer progression and generate a cancer stem cell population. Citation Format: Adam Officer, Farhoud Faraji, Sydney Ramirez, Alon Goren, Pablo Tamayo, J. Silvio Gutkind. YAP-driven epigenetic reprogramming in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1707.

Abstract 614: Src family kinase inhibition demonstrates antitumor activity in vitro and in patient-derived xenograft models of human cholangiocarcinoma

Abstract Background: Cholangiocarcinoma (CCA) is a lethal and heterogenous malignancy of the biliary tree characterized by perineural invasion. About 85% of CCA show activation of YAP signaling, which promotes proliferation, anti-apoptosis, and therapeutic resistance. YAP signaling is mediated by Src family kinases (SFK) by phosphorylation of YAP (pYAPy357). NXP900 is a first-in-class, highly selective, SFK inhibitor with a novel mechanism of action which locks SFK in its closed and inhibited conformation, in contrast to other Src inhibitors such as dasatinib. A phase 1a study of NXP900 in patients with advanced solid tumors was recently initiated. Here, we examined treatment responses of CCA to NXP900 in vitro and in vivo. Methods: Cell viability was determined in seven CCA cell lines by CellTiter-Glo. Cell death and apoptosis were assayed by PI/Hoechst and Caspase-Glo 3/7 assay. CalcuSyn software was used to determine synergistic drug effects. Immunoblot analysis, RT-PCR, and IF staining of YAP localization were used to evaluate YAP knockdown. Clones of human CCA cell lines resistant to NXP900 were generated through escalating exposure to NXP900 over 6 months. Five patient-derived xenograft (PDX) tumors were expanded into flanks of NOD/SCID mice. Tumor bearing mice were randomized 1:1 with 5 mice per arm and treated with vehicle or NXP900 (40 mg/kg) once daily for up to 4 weeks. To determine drivers of sensitivity and resistance, predicted sensitivity scores based on tumor growth were linked to multi-omics (RNA seq and [phospho-]proteomics) of our PDX models. Results: All cell lines were sensitive to NXP900 with IC50 values between 7nM-15µM; IC50’s of resistant clones were 1000 times higher. NXP900 induced more cell death compared to vehicle, which appeared to be through apoptosis. NXP900 inhibited pSrc, decreased pYAPy357, and upregulated inactive pYAPs127. Correspondingly, decreased YAP target gene (Cyr61, NUAK, and CTGF) levels and a nuclear-to-cytoplasmic translocation were observed. NXP900/GemCis combination therapy increased cell death demonstrated a synergistic effect (Combination Indices <1) at all concentrations. In our PDX models, treatment was associated with a significant decrease in tumor growth in 3 models (mean fold change 3.1 vs. 14.2; 1.0 vs. 7.6; 1.4 vs. 2.6). The major resistant signature was the TRK signaling network, involved in nerve growth factor binding activity, while drivers of sensitivity included the SRC network. Conclusion: NXP900 demonstrated therapeutic activity in vitro and in human PDX models. We are currently performing multi-omic approaches in NXP900 sensitive and resistant cell lines to unravel determinants of activity and resistance. Additional in vivo studies will be performed to determine effects of NXP900/GemCis and NXP900/anti-PDL-1 combination therapy. Citation Format: Hendrien Kuipers, Jennifer L. Tomlinson, Danielle M. Carlson, Amro M. Abdelrahman, Erik Jessen, Jack W. Sample, Nathan W. Werneburg, Hannah E. Stumpf, Mark J. Truty, Sumera I. Ilyas, Gregory J. Gores, Rory L. Smoot. Src family kinase inhibition demonstrates antitumor activity in vitro and in patient-derived xenograft models of human cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 614.

Phase separation of YAP-MAML2 differentially regulates the transcriptome

Phase separation (PS) drives the formation of biomolecular condensates that are emerging biological structures involved in diverse cellular processes. Recent studies have unveiled PS-induced formation of several transcriptional factor (TF) condensates that are transcriptionally active, but how strongly PS promotes gene activation remains unclear. Here, we show that the oncogenic TF fusion Yes-associated protein 1-Mastermind like transcriptional coactivator 2 (YAP-MAML2) undergoes PS and forms liquid-like condensates that bear the hallmarks of transcriptional activity. Furthermore, we examined the contribution of PS to YAP-MAML2-mediated gene expression by developing a chemogenetic tool that dissolves TF condensates, allowing us to compare phase-separated and non-phase-separated conditions at identical YAP-MAML2 protein levels. We found that a small fraction of YAP-MAML2-regulated genes is further affected by PS, which include the canonical YAP target genes CTGF and CYR61, and other oncogenes. On the other hand, majority of YAP-MAML2-regulated genes are not affected by PS, highlighting that transcription can be activated effectively by diffuse complexes of TFs with the transcriptional machinery. Our work opens new directions in understanding the role of PS in selective modulation of gene expression, suggesting differential roles of PS in biological processes.

Blood Flow Directs Yap/Taz-Mediated Transcriptional Regulation of Self-Renewal Programs to Control Developmental HSPC Expansion By Mechanical Stimulation of Piezo1

Hematopoietic stem and progenitor cells (HSPCs) emerge from artery-derived hemogenic endothelium (HE) in the vertebrate embryo during development. This process entails a cellular reprogramming from endothelial to hematopoietic gene signatures driven by the Runx1 transcription factor (TF), which is referred to as the endothelial-to-hematopoietic transition (EHT). A major goal of cellular therapeutics is to derive patient-specific HSPCs from iPSCs for clinical use, yet current differentiation protocols largely fail to recapitulate EHT to produce or expand long-lived multi-potent HSPCs in vitro suggesting an incomplete understanding of the in vivo regulatory cues. Physical forces of wall shear stress (WSS) and cyclic stretch (CS) produced by hemodynamic blood flow in embryos are one such cue required during EHT to generate HSPCs from HE, however the mechanisms by which these forces are sensed and converted into a “stemness” regulatory module remain undefined. Our previous work implicated the Hippo pathway TF YAP as essential for the maintenance, but not initiation, of the hematopoietic program in newly specified HE. Here, using scRNA-sequencing of sorted zebrafish trunk endothelial cells to capture all stages of EHT progression, we find that overexpression of YAP drives lipid metabolism, cell cycling, and propagation of a hematopoietic gene regulatory network (GRN) in the earliest specified HE cells. By employing a heat shock-inducible dominant negative YAP zebrafish line, we unmask a latent role for the YAP paralogue TAZ in hematopoiesis, which can promote CD41 + and Flk +/Myb + HSPC production upon reduced YAP function. YAP and TAZ initiate transcriptional responses downstream of mechanical stimuli and require DNA binding cofactors to regulate target genes. Surprisingly, luciferase assays in HEK293 cells demonstrated a potent synergistic effect of TAZ/RUNX1, but not YAP/RUNX1, in transcriptional regulation at RUNX enhancers. Finally, by pharmacologic and genetic manipulation, we identify the stretch-gated membrane ion channel Piezo1 as a potent regulator of CS-induced YAP/TAZ mechanotransduction in HE. Stimulation of zebrafish embryos with the Piezo1 small molecule agonist Yoda1 significantly increases HSPC number and YAP target gene expression in a YAP-dependent fashion. A similar modulation of blood and YAP target genes in human iPSC-derived CD34+ HE cells is seen with Yoda1, suggesting that this stretch-Piezo1-YAP axis can be chemically tuned in vitro to enhance HSPC differentiation. These results illuminate molecular details of how YAP/TAZ directs the production of HSPCs from HE in response to mechanical cues, and have broader implications for alternate regulatory effects of mechanically-stimulated Hippo TFs depending on the transcriptional milieu in cell-type specific contexts.

TMIC-45. SEX DIFFERENCES IN YAP1-CD276 EXPRESSION AND FUNCTION IN MEDULLOBLASTOMA IMMUNE EVASION

Abstract Sex differences in human diseases are well established; however, little is known about the molecules that link oncogenic events and sex as a biological variable in tumorigenesis and treatment response. Here, we report that Yap1 has a sex-specific role in SHH medulloblastoma (MB) progression and immune suppression. Through integrated genomic, transcriptomic, proteomic, and in vivo transcriptional target analyses, we show that Yap1 concurrently promotes stemness and blocks differentiation of cancer stem cells through at least two distinct but complementary molecular mechanisms. In parallel, Yap1 also promotes immune suppressive tumor microenvironment by directly regulating CD276, Csf1, Igf1, and Igfbp3 transcription and modulating the IL6-JAK-STAT3, TNFR1, TGF-β, and CCL5 immune pathways. Notably, while Yap1 function is critical for promoting stemness in male and female MB cells, Yap1 plays a more dominant role in promoting immune suppression and Th2 polarization in male MBs. Furthermore, we discovered that a YAP1-target gene, CD276, expression level is higher in female MBs in both mice and humans, demonstrating for the first-time sex differences in expression levels of an immune checkpoint molecule in MB. Functionally, anti-CD276 treatment is more effective in reversing T cell suppression from male MB cells compared to female MB cells, and CD276 expression level stratifies male but not female MB patient survival. Supporting the clinical relevance of our findings, we show that the sex difference in Yap1 function is evolutionarily conserved across multiple tumor types: YAP1 target gene signature predicts poor survival in males but not females in MB, glioblastoma, low-grade glioma, mesothelioma, and hepatocellular carcinoma. In summary, we provide the first evidence of sex-biased expression and function of YAP1 and CD276 in mouse and human cancers and identified the YAP1-CD276 axis as a key contributor to sex differences in immune checkpoint response.

Chlamydial YAP activation in host endocervical epithelial cells mediates pro-fibrotic paracrine stimulation of fibroblasts.

Chronic or repeated infection of the female upper genital tract by C. trachomatis can lead to severe fibrotic sequelae, including tubal factor infertility and ectopic pregnancy. However, the molecular mechanisms underlying this effect are unclear. In this report, we define a transcriptional program specific to C. trachomatis infection of the upper genital tract, identifying tissue-specific induction of host YAP-a pro-fibrotic transcriptional cofactor-as a potential driver of infection-mediated fibrotic gene expression. Furthermore, we show that infected endocervical epithelial cells stimulate collagen production by fibroblasts and implicate chlamydial induction of YAP in this effect. Our results define a mechanism by which infection mediates tissue-level fibrotic pathology via paracrine signaling and identify YAP as a potential therapeutic target for the prevention of Chlamydia-associated scarring of the female genital tract.

Identification of the primary ciliary proteins IFT38 and IFT144 to enhance serum-mediated YAP activation and cell proliferation

Primary cilia are essential cellular antennae that transmit external signals into intracellular responses. These sensory organelles perform crucial tasks in triggering intracellular signaling pathways, including those initiated by G protein-coupled receptors (GPCRs). Given the involvement of GPCRs in serum-induced signaling, we investigated the contribution of ciliary proteins in mitogen perception and cell proliferation. We found that depletion of cilia via IFT88 silencing impaired cell growth and repressed YAP activation against serum and its mitogenic constituents, namely lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P). To identify the key player of serum mitogen signaling, a mutant cell line library with 30 ablated individual ciliary proteins was established and screened based on YAP dephosphorylation and target gene induction. While 9 of them had altered signaling, ablation of IFT38 or IFT144 led to a particularly robust repression of YAP activation upon LPA and S1P. The deficiency of IFT38 and IFT144 attenuated cell proliferation, as corroborated in either 2-dimensional cultures or tumor spheroids. In subcutaneous skin melanoma patients, expression of IFT38 and IFT144 was associated with unfavorable outcomes in overall survival. In conclusion, our study demonstrates the involvement of ciliary proteins in mitogen signaling and identifies the regulatory roles of IFT38 and IFT144 in serum-mediated Hippo pathway signaling and cellular growth.

AMPK-Dependent YAP Inhibition Mediates the Protective Effect of Metformin against Obesity-Associated Endothelial Dysfunction and Inflammation.

Hyperglycemia is a crucial risk factor for cardiovascular diseases. Chronic inflammation is a central characteristic of obesity, leading to many of its complications. Recent studies have shown that high glucose activates Yes-associated protein 1 (YAP) by suppressing AMPK activity in breast cancer cells. Metformin is a commonly prescribed anti-diabetic drug best known for its AMPK-activating effect. However, the role of YAP in the vasoprotective effect of metformin in diabetic endothelial cell dysfunction is still unknown. The present study aimed to investigate whether YAP activation plays a role in obesity-associated endothelial dysfunction and inflammation and examine whether the vasoprotective effect of metformin is related to YAP inhibition. Reanalysis of the clinical sequencing data revealed YAP signaling, and the YAP target genes CTGF and CYR61 were upregulated in aortic endothelial cells and retinal fibrovascular membranes from diabetic patients. YAP overexpression impaired endothelium-dependent relaxations (EDRs) in isolated mouse aortas and increased the expression of YAP target genes and inflammatory markers in human umbilical vein endothelial cells (HUVECs). High glucose-activated YAP in HUVECs and aortas was accompanied by increased production of oxygen-reactive species. AMPK inhibition was found to induce YAP activation, resulting in increased JNK activity. Metformin activated AMPK and promoted YAP phosphorylation, ultimately improving EDRs and suppressing the JNK activity. Targeting the AMPK-YAP-JNK axis could become a therapeutic strategy for alleviating vascular dysfunction in obesity and diabetes.