YAP1 Deletion Research Articles

YAP1 preserves tubular mitochondrial quality control to mitigate diabetic kidney disease

Renal tubule cells act as a primary site of injury in diabetic kidney disease (DKD), with dysfunctional mitochondrial quality control (MQC) closely associated with progressive kidney dysfunction in this context. Our investigation delves into the observed inactivation of yes-associated protein 1 (YAP1) and consequential dysregulation of MQC within renal tubule cells among DKD subjects through bioinformatic analysis of transcriptomics data from the Gene Expression Omnibus (GEO) dataset. Receiver operating characteristic curve analysis unequivocally underscores the robust diagnostic accuracy of YAP1 and MQC-related genes for DKD. Furthermore, we observed YAP1 inactivation, accompanied by perturbed MQC, within cultured tubule cells exposed to high glucose (HG) and palmitic acid (PA). This pattern was also evident in the tubulointerstitial compartment of kidney sections from biopsy-approved DKD patients. Additionally, renal tubule cell-specific Yap1 deletion exacerbated kidney injury in diabetic mice. Mechanistically, Yap1 deletion disrupted MQC, leading to mitochondrial aberrations in mitobiogenesis and mitophagy within tubule cells, ultimately culminating in histologic tubular injury. Notably, Yap1 deletion-induced renal tubule injury promoted the secretion of C-X-C motif chemokine ligand 1 (CXCL1), potentially augmenting M1 macrophage infiltration within the renal microenvironment. These multifaceted events were significantly ameliorated by administrating the YAP1 activator XMU-MP-1 in DKD mice. Consistently, bioinformatic analysis of transcriptomics data from the GEO dataset revealed a noteworthy upregulation of tubule cells-derived chemokine CXCL1 associated with macrophage infiltration among DKD patients. Crucially, overexpression of YAP1 via adenovirus transfection sustained mitochondrial membrane potential, mtDNA copy number, oxygen consumption rate, and activity of mitochondrial respiratory chain complex, but attenuated mitochondrial ROS production, thereby maintaining MQC and subsequently suppressing CXCL1 generation within cultured tubule cells exposed to HG and PA. Collectively, our study establishes a pivotal role of tubule YAP1 inactivation-mediated MQC dysfunction in driving DKD progression, at least in part, facilitated by promoting M1 macrophage polarization through a paracrine-dependent mechanism.

IMMU-64. SEX-SPECIFIC ROLES OFYAP1 AND CD276/B7-H3 IN MEDULLOBLASTOMA IMMUNE SUPPRESSION

Abstract The molecular mechanism governing sex disparities in cancer prevalence, progression, and treatment outcomes represents an important yet understudied area of research with significant implications for cancer treatment. Medulloblastoma (MB), the most prevalent pediatric brain malignancy, exhibits a notable sex bias in incidence and survival rates. Males have a higher incidence rate across all age groups and have worse prognoses than their females for age groups over three years old. Here, we report an unanticipated sex-biased role of the oncogene Yap1 in SHH medulloblastoma. Our results reveal that Yap1 deletion significantly prolongs survival in male, but not female mice with SmoM2-driven SHH MB. Employing an integrated multi-omics approach, we demonstrate that YAP1 is required to maintain cancer stem cells simultaneously activating stemness genes (such as Sox2) and repressing differentiation genes (such as Neurod1 and Zic1/2) in both sexes. In contrast, Yap1 plays a more critical role in immune evasion in males than in females. Specifically, YAP1 regulates the expression of the immune checkpoint molecule Cd276/B7-H3, which suppresses cytotoxic T cell function. In males, CD276 blockade or Yap1 deletion in MB cells is enough to reverse T cell suppression effectively. In females, neither CD276 blockade nor Yap1 deletion alone in MB cells can reverse T cell suppression. Furthermore, YAP1’s direct transcriptional target gene signatures, including CD276, predict survival across multiple human cancers in male patients. These findings suggest broader implications of our findings beyond medulloblastoma and indicate a highly conserved mechanism across species. Our study provides compelling evidence for the male-biased efficacy of Yap1 or CD276 inhibitors and presents novel insights into sex-specific mechanisms of cancer immune evasion.

A Retinoic Acid:YAP1 signaling axis controls atrial lineage commitment.

Vitamin A/Retinoic Acid (Vit A/RA) signaling is essential for heart development. In cardiac progenitor cells (CPCs), RA signaling induces the expression of atrial lineage genes while repressing ventricular genes, thereby promoting the acquisition of an atrial cardiomyocyte cell fate. To achieve this, RA coordinates a complex regulatory network of downstream effectors that is not fully identified. To address this gap, we applied a functional genomics approach (i.e scRNAseq and snATACseq) to untreated and RA-treated human embryonic stem cells (hESCs)-derived CPCs. Unbiased analysis revealed that the Hippo effectors YAP1 and TEAD4 are integrated with the atrial transcription factor enhancer network, and that YAP1 is necessary for activation of RA-enhancers in CPCs. Furthermore, in vivo analysis of control and conditionally YAP1 KO mouse embryos (Sox2-cre) revealed that the expression of atrial lineage genes, such as NR2F2, is compromised by YAP1 deletion in the CPCs of the second heart field. Accordingly, we found that YAP1 is required for the formation of an atrial chamber but is dispensable for the formation of a ventricle, in hESC-derived patterned cardiac organoids. Overall, our findings revealed that YAP1 is a non-canonical effector of RA signaling essential for the acquisition of atrial lineages during cardiogenesis.

Abstract 3037: Sex-specific differences in Yap1 and Cd276 function and expression regulate medulloblastoma progression and immune evasion

Abstract Unveiling the molecular mechanisms underlying sex-specific differences in cancer initiation, progression and treatment outcomes will provide novel insights that will advance cancer research and clinical care. This study highlights an unexpected, sex-biased role of the oncogene Yap1 in SHH medulloblastoma (MB). We discovered that Yap1 deletion in SmoM2-driven SHH MB significantly prolongs survival in male but not female mice. Using an integrated multi-omics approach, we show that YAP1 promotes cancer stem cell maintenance through concurrent transcriptional activation of stemness genes, such as Sox2, and repression of differentiation genes such as NeuroD1 and Zic1/2. Interestingly, while Yap1 is essential for maintaining cancer stem cells in both sexes, it plays a more critical role in immune evasion in males. Specifically, YAP1 regulates the expression of an immune checkpoint molecule Cd276 to suppress T cell function. Blocking CD276 or deleting Yap1 is sufficient to significantly reverse T cell suppression in males but not females. Furthermore, YAP1 direct targets of transcriptional regulation, including CD276, predicts survival in male but not female patients across multiple human cancers, suggesting that our findings have broader implications beyond medulloblastomas and is conserved across species. This study provides compelling evidence for male-biased susceptibility to Yap1 inhibition and uncovers the YAP1-CD276 axis as a sexually diverse pathway in T cell suppression in tumors. Citation Format: Nourhan Abdelfattah, Sivaraman Natarajan, Han Nhat Tran, Jose Maldonado, Rachael McMinimy, Hannah Borland, Shu-hsia Chen, Fernando Camargo, James Olson, Joshy George, Kyuson Yun. Sex-specific differences in Yap1 and Cd276 function and expression regulate medulloblastoma progression and immune evasion [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 3037.

The APC/C Activator Cdh1p Plays a Role in Mitochondrial Metabolic Remodelling in Yeast

Cdh1p is one of the two substrate adaptor proteins of the anaphase promoting complex/cyclosome (APC/C), a ubiquitin ligase that regulates proteolysis during cell cycle. In this work, using a proteomic approach, we found 135 mitochondrial proteins whose abundance was significantly altered in the cdh1Δ mutant, with 43 up-regulated proteins and 92 down-regulated proteins. The group of significantly up-regulated proteins included subunits of the mitochondrial respiratory chain, enzymes from the tricarboxylic acid cycle and regulators of mitochondrial organization, suggesting a metabolic remodelling towards an increase in mitochondrial respiration. In accordance, mitochondrial oxygen consumption and Cytochrome c oxidase activity increased in Cdh1p-deficient cells. These effects seem to be mediated by the transcriptional activator Yap1p, a major regulator of the yeast oxidative stress response. YAP1 deletion suppressed the increased Cyc1p levels and mitochondrial respiration in cdh1Δ cells. In agreement, Yap1p is transcriptionally more active in cdh1Δ cells and responsible for the higher oxidative stress tolerance of cdh1Δ mutant cells. Overall, our results unveil a new role for APC/C-Cdh1p in the regulation of the mitochondrial metabolic remodelling through Yap1p activity.

Abstract PR001: Oncogene-induced matrix reorganization controls CD8+ T cell immunity in the UPS microenvironment

Abstract CD8+ T cell dysfunction, characterized by reduced effector function, impaired proliferation, and inhibitory receptor upregulation, is a fundamental barrier to anti-tumor immunity. However, molecular mechanisms underlying the regulation of CD8+ T cell dysfunction in the tumor microenvironment (TME) are incompletely understood. In solid cancers, the extracellular matrix (ECM) facilitates tumor progression in part by inhibiting T cell migration/infiltration, but the impact of individual tumor-associated ECM molecules on T cell function remains unclear. Therefore, we investigated the regulation and impact of ECM composition on CD8+ T cell function in muscle-derived undifferentiated pleomorphic sarcoma (UPS). UPS exhibits durable responses to checkpoint therapy in a subset of human patients, potentially offering valuable insights into strategies for ameliorating T cell function and improving patient responses to immunotherapy. Using the autochthonous Kras G12D/+; Trp53 fl/fl (KP) murine model of UPS, we previously showed that deletion of the central Hippo pathway effector Yap1 (Kras G12D/+; Trp53 fl/fl; Yap1 fl/fl; KPY) suppressed UPS cell proliferation and tumor progression. Given the well-established role of Yap1 in mechanotransduction, we leveraged this system to investigate the effects of Yap1 on the ECM and CD8+ T cell function in UPS. We discovered that loss of UPS-cell intrinsic Yap1 reduced the proportion of dysfunctional CD8+ T cells in the TME and enhanced T cell cytolytic capacity. Yap1 loss also downregulated expression of multiple collagen genes in UPS cells and bulk tumors, including those that encode collagen type VI (ColVI). ColVI is a beaded microfilament collagen that binds to fibril-forming collagens in the ECM, such as collagen type I (ColI), and has been implicated in the pathogenesis of skeletal muscle myopathies. These data suggest that proper ColVI structure and function are critical for normal skeletal muscle physiology, with important implications for muscle-derived tumors such as UPS. Accordingly, COL6A1 was upregulated in human UPS relative to normal skeletal muscle, and inversely associated with UPS patient survival. Moreover, loss of UPS cell-intrinsic Col6a1 suppressed tumor progression, enhanced T cell cytolytic function, and attenuated CD8+ T cell exhaustion, phenocopying the effects of Yap1 deletion. Mechanistically, Yap1-mediated ColVI deposition promoted CD8+ T cell dysfunction by remodeling ColI networks in the UPS TME, and inhibiting T cell autophagic flux. Furthermore, ColI depletion dramatically increased tumor growth in an immunocompetent setting. Our findings reveal a novel role for UPS cell-intrinsic Yap1 in immune activation, and demonstrate that ColVI and ColI have opposing functions downstream of Yap in this context. These results underscore the need to systematically evaluate the roles of individual ECM components in the regulation of immune cell function, and implicate YAP1 and/or COLVI targeting as potential strategies for improving the efficacy of immunotherapy in human patients. Citation Format: Ashley M. Fuller, Hawley C. Pruitt, Hoogeun Song, Ying Liu, Ann Devine, Rohan S. Katti, Samir Devalaraja, Gabrielle E. Ciotti, Michael Gonzalez, Erik F. Williams, Ileana Murazzi, Nicolas Skuli, Hakon Hakonarson, Kristy Weber, Malay Haldar, Joseph A. Fraietta, Sharon Gerecht, T. S. Karin Eisinger-Mathason. Oncogene-induced matrix reorganization controls CD8+ T cell immunity in the UPS microenvironment [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr PR001.

Therapeutic implication of SOX9 and YAP1 co‐repression in advanced intrahepatic cholangiocarcinoma

IntroductionIntrahepatic cholangiocarcinoma (iCCA) is a liver tumor of increasing incidence and devastating prognosis. The most critical barrier for developing a globally effective therapy for iCCA is molecular heterogeneity caused by diverse molecular drivers and cellular origins in the liver. Clinically, over 90% of iCCAs show alterations of YAP1 and/or SOX9. Therefore, we investigated the therapeutic potential of co‐repression of YAP1 and SOX9 as a wide range therapeutic option for advanced iCCA.MethodsBy immunohistochemistry (IHC) using 2 tissue microarrays, we examined the localization of SOX9 and YAP in clinical iCCA. We also assessed clinical relevance of AKT‐NICD (AN) /YAP (AY)‐dependent hepatocyte‐driven iCCA by bioinformatic analysis comparing their transcriptome with large human datasets. Lastly, we comprehensively investigated therapeutic effect of simultaneous deletion of Sox9 and Yap1 in advanced murine iCCA using respective floxed mice.ResultsOver 90% of human CCA samples (n=108) exhibited nuclear SOX9 and YAP1. We also identified significant upregulation of p‐AKT, SOX9 and YAP1 in HCs of patients with primary sclerosing cholangitis and nonalcoholic steatotis hepatitis, well‐known risk factors for iCCA. While co‐expression of AN led to hepatocyte‐derived lethal iCCA, conditional deletion of either Yap1 or Sox9 dramatically reduced AN‐ iCCA development. Yap1 deletion impaired HC‐to‐biliary epithelial cell (BEC) fate conversion and tumor cell proliferation, while Sox9 elimination only repressed proliferation. Interestingly, following single deletion of either Yap1 or Sox9 we observed a few iCCA tumors expressing either SOX9 or YAP1 but not both. This is also identified in a small subset of human iCCA; SOX9+/YAP1‐ (4.6%) or SOX9‐/YAP1+ (3.7%), suggesting that single deletion of YAP1 or SOX9 is not sufficient to abrogate clinical iCCA. We also found that simultaneous deletion of Yap1 and Sox9 completely prevents iCCA development. Remarkably, co‐deletion of Yap1 and Sox9profoundly decreases fully developed lethal iCCA burden while extending survival. However, singular deletion of either shows no survival benefit compared to control group harboring advanced iCCA as a therapeutic manner.ConclusionsWe elucidate the critical, but distinct roles of Yap1and Sox9 in iCCA development and demonstrate the therapeutic potential of simultaneous targeting both for wide range of iCCA irrespective of drivers. Ongoing work will comprehensively delineate the molecular mechanism how YAP1 and SOX9 compensate each other when either is removed in iCCA, and will be relevant for studying adaptive resistance against the singular targeting of SOX9 or YAP1 in iCCA.

EMBR-33. YAP1 FUNCTION IN SHH MEDULLOBLASTOMA PROGRESSION AND IMMUNE EVASION

Our incomplete understanding of the key players in Medulloblastoma (MB) development and progression, and their roles in modulating highly Immune desert-like microenvironment in MBs present major hurdles in successfully applying existing therapies and developing new therapies for MBs. Here, we demonstrate that Yap1 acts as a critical modulator of SHH MB (fSmoM2; GFAPcre (SG) and Ptch;p53 SHH-MB mouse models) progression and immune evasion. Yap1 genetic deletion in SG mice significantly extends survival and normalizes brain development by increasing neuronal differentiation. Both bulk and single-cell RNA sequencing analyses show that Yap1 deleted tumors contain cells with more differentiated molecular signatures similar to late CGNPs and differentiating neurons, and less stem-like cells, compared to SG tumors. Additionally, integrated analyses of ChiPseq, RNAseq, and scRNAq data suggest that Yap1 directly binds to the Super enhancer region containing Sox2 and promotes Sox2 expression in SHH MB cells. We postulate that Yap1 expression is maintained or re-activated in SHH MB cells to generate long-term self-renewing tumor cells. Consistently, Yap1-deleted SHH MB or Verteporfin (a small molecule inhibitor of Yap1) -treated Ptch;p53 MB cells lose self-renewal ability in vitro. Furthermore, we hypothesize that a molecular mechanism underlying this stemness promoting function is mediated through Sox2 expression. Intriguingly, Yap1 deletion in SG MBs is accompanied by a significant change in the immune microenvironment, when compared to age-matched SG MBs. There is a significant increase in the number of bone marrow-derived immune cells (including cytotoxic T-cells, neutrophils, and macrophages). RNAseq analysis of rescued tumors shows marked enrichment of interferon-gamma response genes and pro-inflammatory cytokines. This study highlights Yap1 as a crucial mediator of MB progression and a molecular regulator of inflammatory immune cell infiltration into SHH MBs. Consequently, our work paves the way for improving immunotherapy treatments in brain malignancies.

Novel Model of Bile Duct Paucity Demonstrates the Critical Role of Yap1 in Biliary Morphogenesis in Development and Regeneration

BackgroundYes‐associated protein 1 (Yap1) is a critical regulator of liver embryonic development, regeneration and tumorigenesis. Previous studies have shown that Yap1 regulates bile duct formation and hepatocyte transdifferentiation to cholangiocytes, but the mechanisms by which Yap1 regulates biliary morphogenesis as well as the functions of Yap1 in early embryonic liver development remain unknown.MethodsTo elucidate the role of Yap1 in early liver development, we developed a novel mouse model (Yap1 KO) in which Yap1 is deleted from the foregut endoderm progenitors by E12.5 using the Foxa3 promoter to drive Cre recombinase expression. Here, we comprehensively characterize the Yap1 KO mice through histology, whole organ tissue clearing and confocal microscopy and additional functional techniques.ResultsYap1 KO mice lack Yap1 in the liver from early development. Strong nuclear expression of Yap1 that was seen in hepatoblasts in WT livers at E14.5 was clearly absent in the KO mice. Although Yap1 KO mice were viable and have fully formed liver and digestive organs, they exhibited jaundice and stunted growth. Histological analysis identified a failure of intrahepatic bile duct formation. Yap1 deletion did not block Notch activation as shown by the appearance of Hes1‐positive and Sox9‐positive ductal plate cells in both WT and KO mice. Rather, Yap1 deletion prevented the morphological assembly of a two‐cell layer with a lumen from an asymmetrical single‐layered ductal plate. Surprisingly, KO mice survive long‐term with normalization of serum liver enzymes despite persistent cholestatic injury, suggesting hepatocyte adaptation to injury. While a recent study of Notch‐deficient livers (a model of Alagille syndrome) described complete de novo hepatocyte‐derived generation of bile ducts by 4 months of age, Yap1‐deficient livers fail to regenerate bile ducts by 8 months of age. Liver tissue clearing and 3D whole liver immunofluorescence imaging showed severely abbreviated biliary tree in KO mice relative to WT. Notably, we observe the appearance of a ductular reaction in the hilar region of the liver consisting of Yap1‐positive ducts surrounded by fibrosis and inflammation. Although all hepatocytes remain Yap1‐negative, the extrahepatic biliary tree remains unaffected by the Cre and is Yap1‐positive, suggesting a potential source of Yap1‐positive ductular cells.ConclusionsOur results demonstrate that Yap1 is dispensable for early development of the liver and foregut but is critical for biliary morphogenesis. We further show absence of hepatocyte‐to‐cholangiocyte transdifferentiation in Yap1 KO. Thus, Yap1 is essential for biliary morphogenesis regardless of the cellular origin, whether from hepatoblasts in development or from hepatocytes in liver regeneration.Support or Funding InformationThis work was supported by NIH grants 1R01DK62277, 1R01DK100287, 1R01DK116993 and Endowed Chair for Experimental Pathology to S.P.M, 1P30DK120531‐01 to the Pittsburgh Liver Research Center, and T32EB0010216 to LM.

YAP1 Mediates Resistance to MEK1/2 Inhibition in Neuroblastomas with Hyperactivated RAS Signaling.

Relapsed neuroblastomas are enriched with activating mutations of the RAS-MAPK signaling pathway. The MEK1/2 inhibitor trametinib delays tumor growth but does not sustain regression in neuroblastoma preclinical models. Recent studies have implicated the Hippo pathway transcriptional coactivator protein YAP1 as an additional driver of relapsed neuroblastomas, as well as a mediator of trametinib resistance in other cancers. Here, we used a highly annotated set of high-risk neuroblastoma cellular models to modulate YAP1 expression and RAS pathway activation to test whether increased YAP1 transcriptional activity is a mechanism of MEK1/2 inhibition resistance in RAS-driven neuroblastomas. In NLF (biallelic NF1 inactivation) and SK-N-AS (NRAS Q61K) cell lines, trametinib caused a near-complete translocation of YAP1 protein into the nucleus. YAP1 depletion sensitized neuroblastoma cells to trametinib, while overexpression of constitutively active YAP1 protein induced trametinib resistance. Mechanistically, significant enhancement of G1-S cell-cycle arrest, mediated by depletion of MYC/MYCN and E2F transcriptional output, sensitized RAS-driven neuroblastomas to trametinib following YAP1 deletion. These findings underscore the importance of YAP activity in response to trametinib in RAS-driven neuroblastomas, as well as the potential for targeting YAP in a trametinib combination. SIGNIFICANCE: High-risk neuroblastomas with hyperactivated RAS signaling escape the selective pressure of MEK inhibition via YAP1-mediated transcriptional reprogramming and may be sensitive to combination therapies targeting both YAP1 and MEK.

Lawsone, a 2-hydroxy-1,4-naphthoquinone from Lawsonia inermis (henna), produces mitochondrial dysfunctions and triggers mitophagy in Saccharomyces cerevisiae.

Lawsone is a natural naphthoquinone present in the henna leaf extract with several cytotoxic activities and used as precursor for synthesis of various pharmaceutical compounds. Its biological activities are thought to be the result of oxidative stress generated, although the hydroxy group at position C-2 in its structure tends to reduce its electrophilic potential. In view of lack of knowledge on its activity, the present work aimed to elucidate the biological effect of lawsone using the yeast Saccharomyces cerevisiae. In the model strain BY4741 it was defined 229 mmol/L as the minimal inhibitory concentration (MIC). Using 172 mmol/L as sub-MIC value it was observed that yap1 deletion mutant was sensitive to lawsone independent the presence of oxygen. Lawsone affected yeast growth in glycerol, indicating interference in the respiratory metabolism. Intracellular content of thiol groups did not indicate intensive oxidative stress and the presence of the anti-oxidant N-acetylcysteine (NAC) exacerbated lawsone toxicity. By analysing the sensitivity of atg mutant strains and the localization of GFP-Atg8 fusion protein, it was concluded that lawsone primarily produces mitochondrial malfunctioning, leading to indirect oxidative stress. It triggers the autophagic response that ultimately induces mitophagy.

PDTM-09. Yap1 FUNCTION IN SEX-BIASED MEDULLOBLASTOMA FORMATION AND ANTI-TUMOR IMMUNITY

Abstract Immunotherapies offer remarkable potential to provide robust therapeutic benefit. Patients suffering from medulloblastoma (MB), the most frequent pediatric brain malignancy, can especially benefit from this approach, minimizing the devastating side effects of aggressive radiation and chemotherapies that disrupt normal brain development. However, regulators of the immune landscape remain poorly understood and no effective immunotherapies exist yet for MB. Here, we describe a sex-dependent Yap1 function in fSmoM2;GFAPcre SHH-MB (SG) mouse model. We show that Yap1 is both a cell-autonomous regulator of MB stem-cells and a non-cell-autonomous regulator of immune infiltrates in SHH-MB. Yap1 deletion in SG mice results in increased neuronal differentiation, significantly extended survival, and enhanced infiltration of peripheral blood immune cells (including cytotoxic T-cells, neutrophils, and macrophages). Additionally, this rescue phenotype is observed in a sex-biased manner: 65% of Yap1f/f;fSmoM2;GFAPcre males are rescued in contrast to 35% of females. These observations implicate Yap1 as a mediator of sex-biased brain-tumor formation, either through direct modulation of MB cells and/or through indirectly mediating the MB immune landscape. We are currently testing the role of sex-specific differences in the developing mouse brain to elucidate context-dependent function of Yap1 in MB genesis and maintenance.

Abstract B21: YAP1-mediated circadian oscillation in sarcoma

Abstract Soft-tissue sarcomas present a unique challenge to researchers due to their heterogeneity. However, one common mechanism associated with more than 25% of diagnosed sarcomas is deregulation of the Hippo pathway, a critical signaling cascade that responds to cell contact inhibition, among other factors, and negatively regulates cell proliferation. In quiescent cells the pathway remains “on,” which maintains activation of the kinase cascade that phosphorylates and degrades YAP1. However, inactivation of the pathway stabilizes YAP1, allowing it to translocate to the nucleus and activate transcription of pro-proliferative gene targets. Microarray analysis of tumor tissue from the LSLKrasG12D/+;Trp53fl/fl (KP) mouse model of undifferentiated pleomorphic sarcoma (UPS) revealed that YAP1 deletion (KPY) reduces sarcoma cell proliferation and increases expression of circadian rhythm genes. Notably, deletion of YAP1 in KP tumors resulted in a 2.5-fold increase in expression of PER1. Although PER1 has primarily been characterized as a negative regulator of the circadian clock, upregulation of PER1 is known to modulate the G2/M cell cycle checkpoint at both the protein and mRNA levels. Our findings represent a novel link between the circadian clock and Hippo pathway. Our studies also indicate that Hippo signaling is epigenetically regulated in sarcomas. Thus we employed the epigenetic modulating drugs JQ1, a BET inhibitor, and SAHA, an HDAC inhibitor, to regain control of this pathway. Treatment with these inhibitors significantly reduced YAP1 expression, decreased sarcoma growth in vivo and in vitro, and surprisingly increased muscle cell differentiation markers, including the master muscle regulator MyoD. Under JQ1 and SAHA drug treatment, as well as YAP1 shRNA knockdown conditions, we demonstrated that PER1 is significantly increased at both the protein and transcriptional levels in KP tumor derived cells. Together, these results suggest that YAP1 represses PER1 expression in sarcoma, and that epigenetic therapies can cause reexpression of PER1. Another key cellular response to SAHA/JQ1 exposure in sarcoma cells is alteration in NF-κB signaling. NF-κB, a critical signaling cascade for muscle cell progenitor proliferation, oscillates temporally in sarcoma cells treated with SAHA and JQ1. We hypothesize that this oscillation may be disrupted when PER1 is suppressed due to high YAP1 levels in sarcoma leading to uncontrolled proliferation. NF-κB activation enhances proliferation and suppresses differentiation in normal muscle progenitor cells and myoblasts by preventing MyoD upregulation. Importantly, it has been reported that MyoD is a clock-controlled gene. Given this relationship between MyoD and PER1, it is possible that PER1 functions to regulate MyoD through NF-κB in sarcoma and thus alters the balance between cell proliferation and differentiation. Citation Format: Gloria Marino, Shaun Egolf, Shuai Ye, Koreana Pak, Jenn Shah, Adrian Rivera-Reyes, Susan Chor, T. S. Karin Eisinger-Mathason. YAP1-mediated circadian oscillation in sarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B21.

Abstract 3531: YAP1-mediated circadian oscillation in sarcoma

Abstract Soft tissue sarcomas have presented a unique challenge to researchers due to their heterogeneity. One common mechanism underlying more than 25% of diagnosed sarcomas is the deregulation of the Hippo pathway, a critical signaling cascade that responds to cell contact inhibition, among other factors, and negatively regulates cell proliferation. In quiescent cells the pathway remains “on,” which maintains activation of a kinase cascade that phosphorylates and degrades YAP1. However, inactivation of the pathway stabilizes YAP1 allowing it to translocate to the nucleus and activate transcription of pro-proliferative gene targets. Microarray analysis of muscle tissue from the LSL-KrasG12D/+;Trp53fl/fl (KP) mouse model of undifferentiated pleomorphic sarcoma (UPS) revealed that YAP1 deletion (KPY) reduces cell proliferation and increases expression of circadian rhythm genes including PER1. UPS is a commonly diagnosed and aggressive type of muscle-derived sarcoma. The KP model recapitulates human UPS morphologically and histologically, as well as by gene expression profiling. Although PER1 has primarily been characterized as a negative regulator of the circadian clock, upregulation of PER1 is known to modulate the G2/M cell cycle checkpoint at both the protein and mRNA level independent of p53. However, our findings represent a novel link between the circadian clock and the hippo pathway. Notably, deletion of YAP1 in KP tumors leads to a statistically significant 2.5 fold increase in expression of PER1. The study has two specific aims: 1) to identify the YAP1-dependent function of PER1 in sarcoma and 2) determine whether YAP1 directly or indirectly regulates PER1. We have validated the microarray results in KP tumor derived cell lines as well as in KP and KPY tumor tissue. I have also confirmed PER1 suppression in KP cells under YAP1 short hairpin RNA conditions. Our lab has previously observed that treatment with epigenetic modulating drugs JQ1, a BET inhibitor, and SAHA, an HDAC inhibitor, significantly decreased sarcoma growth in vivo and in vitro when administered alone and had an additive effect when combined. These effects can be explained in part by the finding that treatment with these inhibitors significantly reduces YAP1 expression. Under these conditions, as well as YAP1 knockdown conditions, I demonstrated via qRT-PCR and western blot that PER1 is significantly increased at both the protein and transcriptional levels in KP cells. Additionally, preliminary evidence from an MTT proliferation assay showed loss of PER1 increased sarcoma cell proliferation. Further supporting the hypothesis that PER1 modulation impacts sarcoma proliferation, it has been reported that MyoD, the master regulator of muscle cell differentiation, is itself a clock-controlled gene. Together, these results suggest that YAP1 represses PER1 expression in sarcoma, and that epigenetic treatments can cause re-expression of PER1 which functions to inhibit cell proliferation and may promote differentiation. Citation Format: Gloria Marino, Shuai Ye, Koreana Pak, Jennifer Shah, Jason Godfrey, Susan Chor, Shaun Egolf, T.S. Karin Eisinger-Mathason. YAP1-mediated circadian oscillation in sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3531. doi:10.1158/1538-7445.AM2017-3531

Oocyte-expressed yes-associated protein is a key activator of the early zygotic genome in mouse.

In early mammalian embryos, the genome is transcriptionally quiescent until the zygotic genome activation (ZGA) which occurs 2-3 days after fertilization. Despite a long-standing effort, maternal transcription factors regulating this crucial developmental event remain largely elusive. Here, using maternal and paternal mouse models of Yap1 deletion, we show that maternally accumulated yes-associated protein (YAP) in oocyte is essential for ZGA. Maternal Yap1-knockout embryos exhibit a prolonged two-cell stage and develop into the four-cell stage at a much slower pace than the wild-type controls. Transcriptome analyses identify YAP target genes in early blastomeres; two of which, Rpl13 and Rrm2, are required to mediate maternal YAP's effect in conferring developmental competence on preimplantation embryos. Furthermore, the physiological YAP activator, lysophosphatidic acid, can substantially improve early development of wild-type, but not maternal Yap1-knockout embryos in both oviduct and culture. These observations provide insights into the mechanisms of ZGA, and suggest potentials of YAP activators in improving the developmental competence of cultured embryos in assisted human reproduction and animal biotechnology.

Dose-Dependent Protective and Inductive Effects of Xanthohumol on Oxidative DNA Damage in Saccharomyces cerevisiae

The effect of xanthohumol, a prenylflavonoid isolated from the hop plant (Humulus lupulus L.), on Saccharomyces cerevisiae DNA oxidative damage and viability was evaluated. Yeast cultures under oxidative stress, induced by H2O2, displayed stronger growth in the presence of 5 mg/L of xanthohumol than cultures with only H2O2. Likewise, DNA damage assessed by the comet assay was significantly lower in cells co-incubated with xanthohumol and H2O2. Accordingly, fluorescence of dichlorofluorescein in cells treated with H2O2 and xanthohumol was considerably lower than in cells exclusively treated with H2O2, indicative of a reactive oxygen species scavenging mechanism and consequent formation of oxidation products, as detected by mass spectrometry. However, at concentrations above 5 mg/L, xanthohumol elicited an opposite effect, leading to a slower growth rate and significant increase in DNA damage. A yeast yap1 deletion mutant strain sensitive to oxidative stress grew more slowly in the presence of at least 5 mg/L of xanthohumol than cultures of the wild type, suggesting that xanthohumol toxicity is mediated by oxidative stress. This evidence provides further insight into the impact of xanthohumol on yeast cells, supporting dose-dependent antioxidant/antigenotoxic and prooxidant/genotoxic effects.

The Role of the ABL1/YAP1/P73 Axis in Prevention of DNA Damage-Mediated Apoptosis in Multiple Myeloma

AbstractAbstract 725 Background:Genome integrity plays a crucial role in the development of normal plasma cells to eliminate aberrant ones. Multiple myeloma (MM) is a plasma cell malignancy characterized by complex heterogeneous cytogenetic abnormalities. MM cells show constitutive DNA Damage Response (DDR) and activate compensatory mechanisms to prevent DNA-damage mediated apoptosis. Here we define the molecular mechanisms of these protective effects. Methods:A panel of 15 MM cell lines was used. Blood and BM samples from healthy volunteers and MM patients were obtained after informed consent and subjected to Ficoll-Paque density sedimentation to get mononuclear cells (MNCs). Patient MM cells were isolated from BM MNCs by CD138-positive selection. Lentiviral delivery system was used for expression and knock-down of YAP1 in KMS-18, KMS-20, MM.1S and UTMC-2 MM cell lines. The biologic impact of YAP1 phenotype was evaluated using cell growth, viability and apoptosis assays. Results:We confirmed that a wide range of MM patient cells and MM cell lines have markers of constitutive DDR, including phosphorylation of H2A.X, ATM, ATR, Chk2 and Chk1, assessed by western blot analysis and immunofluorescence. However, these MM cells do not show basal level of apoptosis. Specifically, cleaved forms of caspase 3 and PARP are lacking in non-treated cells, and the absence of co-expression of cleaved caspase 3 with phospho-H2A.X by immunofluorescence confirms that phospho-H2A.X positive cells are viable cells. Since DDR is present in both p53-wild-type (wt) and p53-mutated cell lines, we examined whether ABL1/YAP1/p73 axis represents an alternative and crucial pathway to avoid DNA-damage mediated apoptosis. Indeed, ABL1 is up-regulated and predominantly localizes in the nucleus, a potential apoptotic stimulus, in MM cells, as assessed by western blot and immunofluorescence. To define if ABL1 nuclear localization was triggered by DDR, we treated MM cells with a specific ATM inhibitor (Ku55933) and a DNA damaging agent, doxorubicin. Inhibition of DDR in both p53 wt cell lines (MM.1S and H929) and p53 mutated cell lines (UTMC-2, JJN-3 and KMS-20) causes ABL1 cytoplasmic retention, while doxorubicin increases ABL1 nuclear translocation. Co-treatment with doxorubicin and ABL1 inhibitor STI-571 rescues MM cells from doxorubicin-mediated cell death. In particular, apoptotic cells decrease from 47.2% to 21.5% in U266, from 55.3% to 12.4% in MM.1S, and from 57.9% to 19.1% in UTMC-2 cells in response to combination treatment. To delineate the molecular mechanisms whereby MM cells repress ABL1 pro-apoptotic function, we focused on YAP1, a downstream target of the Hippo pathway involved in ABL1 cascade. We explored a large dataset of aCGH data on MM patients and discovered that YAP1 genomic locus (chr. 11q22) is deleted, associated with BIRC2 and BIRC3 in 11% of patients. This genetic abnormality was also found in KMS-18 and KMS-20 cell lines. Although YAP1 expression was normal in peripheral blood MNCs (PBMCs), its expression was decreased in the majority of patient MM cells and MM cell lines regardless of the presence of focal deletion. Importantly, low expression of YAP1 is associated with poor prognosis of MM patients. To further delineate the biologic significance of YAP1 in MM cells, we re-expressed pLENTI4-YAP1-EGFP in MM cell lines with either YAP1 deletion (KMS-18, KMS-20) or YAP1 low expression (MM.1S). We also silenced YAP1 using two different specific shRNAs in UTMC-2 MM cell line. As expected, YAP1 re-expression reduces cellular growth and increases apoptosis in all cell lines tested (25.7%, 37.1% and 32.3% apoptotic KMS-20, KMS-18 and MM.1S cells, respectively), condition that was further enhanced by doxorubicin treatment. Previous studies have shown that P73 is expressed in MM even though at low levels and we here show that they inversely correlate with YAP1 protein expression. Importantly, YAP1 re-expression increases p73 stability and promotes transcription of p73-target genes including BAX, PUMA and p21. In contrast, UTMC-2-YAP1−/− cells show improved survival with lower levels of basal apoptosis and higher resistance to treatment with bortezomib or doxorubicin. Conclusion:YAP1 mediates a strong apoptotic signal for MM cells. Thus, activation and/or overexpression of YAP1 represent a novel therapeutic strategy to improve outcome of patients with MM. Disclosures:Anderson:Celgene, Millennium, BMS, Onyx: Membership on an entity's Board of Directors or advisory committees; Acetylon, Oncopep: Scientific Founder and Scientific Founder, Scientific Founder and Scientific Founder Other.

Inactivation of YAP1 Enhances Sensitivity of the Yeast RNR3-lacZ Genotoxicity Testing System to a Broad Range of DNA-Damaging Agents

Despite the great advances by using microorganism-based genotoxicity testing systems to assess environmental genotoxic compounds, most of them respond poorly, particularly to oxidative agents. In this study, we systematically examined the RNR3-lacZ reporter gene expression in Saccharomyces cerevisiae mutant strains defective in the protection against reactive oxygen species and found that only YAP1 deletion resulted in a significant enhancement in the detection of oxidative damage. To our surprise, YAP1 deletion also caused an increased cellular sensitivity to a variety of DNA damage. This altered sensitivity appears to be independent of oxidative damage because under conditions in which vitamin C treatment rescued oxidative damage, it failed to reverse the phenotypes caused by other types of DNA damage. Furthermore, although inactivation of cell permeability genes enhanced the RNR3-lacZ detection sensitivity particularly to large molecular weight compounds, their effects on small molecular oxidative agents are minimal. Taken together, this study helps to create a hypersensitive genotoxicity testing system to a broad range of DNA-damaging agents by deleting a single yeast gene.

Adaptation to hydrogen peroxide in Saccharomyces cerevisiae: The role of NADPH-generating systems and the SKN7 transcription factor

A total of 286 H 2O 2-sensitive Saccharomyces cerevisiae deletion mutants were screened to identify genes involved in cellular adaptation to H 2O 2 stress. YAP1, SKN7, GAL11, RPE1, TKL1, IDP1, SLA1, and PET8 were important for adaptation to H 2O 2. The mutants were divisible into two groups based on their responses to a brief acute dose of H 2O 2 and to chronic exposure to H 2O 2. Transcription factors Yap1p, Skn7p, and Gal11p were important for both acute and chronic responses to H 2O 2. Yap1p and Skn7p were acting in concert for adaptation, which indicates that upregulation of antioxidant functions rather than generation of NADPH or glutathione is important for adaptation. Deletion of GPX3 and YBP1 involved in sensing H 2O 2 and activating Yap1p affected adaptation but to a lesser extent than YAP1 deletion. NADPH generation was also required for adaptation. RPE1, TKL1, or IDP1 deletants affected in NADPH production were chronically sensitive to H 2O 2 but resistant to an acute dose, and other mutants affected in NADPH generation tested were similarly affected in adaptation. These mutants overproduced reduced glutathione (GSH) but maintained normal cellular redox homeostasis. This overproduction of GSH was not regulated at transcription of the gene encoding γ-glutamylcysteine synthetase.

AnUstilago maydisGene Involved in H2O2 Detoxification Is Required for Virulence

The fungus Ustilago maydis is a biotrophic pathogen of maize (Zea mays). In its genome we have identified an ortholog of YAP1 (for Yeast AP-1-like) from Saccharomyces cerevisae that regulates the oxidative stress response in this organism. yap1 mutants of U. maydis displayed higher sensitivity to H(2)O(2) than wild-type cells, and their virulence was significantly reduced. U. maydis yap1 could partially complement the H(2)O(2) sensitivity of a yap1 deletion mutant of S. cerevisiae, and a Yap1-green fluorescent protein fusion protein showed nuclear localization after H(2)O(2) treatment, suggesting that Yap1 in U. maydis functions as a redox sensor. Mutations in two Cys residues prevented accumulation in the nucleus, and the respective mutant strains showed the same virulence phenotype as Deltayap1 mutants. Diamino benzidine staining revealed an accumulation of H(2)O(2) around yap1 mutant hyphae, which was absent in the wild type. Inhibition of the plant NADPH oxidase prevented this accumulation and restored virulence. During the infection, Yap1 showed nuclear localization after penetration up to 2 to 3 d after infection. Through array analysis, a large set of Yap1-regulated genes were identified and these included two peroxidase genes. Deletion mutants of these genes were attenuated in virulence. These results suggest that U. maydis is using its Yap1-controlled H(2)O(2) detoxification system for coping with early plant defense responses.