Transcription Factor Yap1 Research Articles

TMIC-67. EMERGING ROLE OF EXTRACELLULAR MATRIX STIFFNESS AS A HALLMARK OF GLIOBLASTOMA: INSIGHT TO MECHANICAL MEMORY FORMATION AND PROLONGED INVASION IN THE TUMOR PERIPHERY

Abstract The extracellular matrix (ECM) plays a critical role in cancer progression, with its biophysical properties influencing cancer behavior. ECM stiffness can induce long-lasting adaptations in cancer cells, promoting sustained invasion through a process known as “mechanical memory” (MM). Glioblastoma (GBM) exhibits spatial heterogeneity in stiffness, with stiffer ECM found at the tumor periphery where invasive cells reside and promote recurrence. We hypothesize that increased stiffness at the tumor periphery primes GBM cells for sustained invasion through MM. METHODS Cell lines were established from image-guided human GBM samples from tumor margin and core (n=10 patients, 20 cell lines). Cells from random locations were conditioned in physiologically relevant extracellular matrices for two weeks. Human organotypic assay (n=3), Migration Assays, Atomic Force Microscopy (AFM), Immunocytochemistry, Western Blot, and RNA-sequencing (n=6) were performed. RESULTS GBM cells at higher stiff surfaces displayed increased migration (p<0.01). Magnetic Resonance Elastography (n=9) confirmed GBM heterogeneity, with softer cores (0.2-0.5kPa) transitioning to stiffer margins (>2kPa). Organotypic scaffolds mimicking the tumor margin promoted enhanced migration of GFP-labeled GBM cells. Margin cells and cells conditioned on stiffer matrices demonstrated higher migratory potential compared to their core and soft-conditioned counterparts (p<0.05). These trends persist over time and across multiple passages. ECM stiffness exposure causes nanomechanical, cytoskeletal, and transcriptional adaptations. AFM shows that cells from the margin (n=6) are softer (p<0.01) and more deformable (p<0.01). RNAseq identified upregulation of MM-specific genes in GBM margin cells. The mechanosensitive transcription factor YAP1 emerged as a key driver of MM. Notably, YAP1 is targetable by the FDA-approved drug Verteporfin. YAP1 expression was elevated in margin and stiff-conditioned cells (p<0.05). Stiff-conditioned cells and margin cells displayed increased sensitivity to Verteporfin treatment (p<0.05), with treatment significantly reducing margin migration compared to the core (p<0.001). CONCLUSION Stiffer GBM margins promote the formation of aggressive phenotypes through MM. Targeting YAP1 with Verteporfin, a readily available drug, presents a promising strategy to halt GBM invasion and improve patient outcomes.

The expression of YAP1 and other transcription factors contributes to lineage plasticity in combined small cell lung carcinoma.

Lineage plasticity in small cell lung carcinoma (SCLC) causes therapeutic difficulties. This study aimed to investigate the pathological findings of plasticity in SCLC, focusing on combined SCLC, and elucidate the involvement of YAP1 and other transcription factors. We analysed 100 surgically resected SCLCs through detailed morphological observations and immunohistochemistry for YAP1 and other transcription factors. Component-by-component next-generation sequencing (n = 15 pairs) and immunohistochemistry (n = 35 pairs) were performed on the combined SCLCs. Compared with pure SCLCs (n = 65), combined SCLCs (n = 35) showed a significantly larger size, higher expression of NEUROD1, and higher frequency of double-positive transcription factors (p = 0.0009, 0.04, and 0.019, respectively). Notably, 34% of the combined SCLCs showed morphological mosaic patterns with unclear boundaries between the SCLC and its partner. Combined SCLCs not only had unique histotypes as partners but also represented different lineage plasticity within the partner. NEUROD1-dominant combined SCLCs had a significantly higher proportion of adenocarcinomas as partners, whereas POU2F3-dominant combined SCLCs had a significantly higher proportion of squamous cell carcinomas as partners (p = 0.006 and p = 0.0006, respectively). YAP1 expression in SCLC components was found in 80% of combined SCLCs and 62% of pure SCLCs, often showing mosaic-like expression. Among the combined SCLCs with component-specific analysis, the identical TP53 mutation was found in 10 pairs, and the identical Rb1 abnormality was found in 2 pairs. On immunohistochemistry, the same abnormal p53 pattern was found in 34 pairs, and Rb1 loss was found in 24 pairs. In conclusion, combined SCLC shows a variety of pathological plasticity. Although combined SCLC is more plastic than pure SCLC, pure SCLC is also a phenotypically plastic tumour. The morphological mosaic pattern and YAP1 mosaic-like expression may represent ongoing lineage plasticity. This study also identified the relationship between transcription factors and partners in combined SCLC. Transcription factors may be involved in differentiating specific cell lineages beyond just 'neuroendocrine'.

WWC1 upregulation accelerates hyperuricemia by reduction in renal uric acid excretion through Hippo signaling pathway

Hyperuricemia (HUA) is a metabolic disorder characterized by elevated serum uric acid (UA), primarily attributed to the hepatic overproduction and renal underexcretion of UA. Despite the elucidation of molecular pathways associated with this underexcretion, the etiology of HUA remains largely unknown. In our study, using by Uox knockout rats, HUA mouse and cell line models, we discovered that the increased WWC1 levels were associated with decreased renal UA excretion. Additionally, using by knockdown and overexpression approaches, we found that WWC1 inhibited UA excretion in renal tubular epithelial cells. Mechanistically, WWC1 activated the Hippo pathway, leading to phosphorylation and subsequent degradation of the downstream transcription factor YAP1, thereby impairing the ABCG2 and OAT3 expression through transcriptional regulation. Consequently, this reduction leaded to a decrease in UA excretion in renal tubular epithelial cells. In conclusion, our study has elucidated the role of upregulated WWC1 in renal tubular epithelial cells inhibiting the excretion of UA in the kidneys and causing HUA.

Bee venom genotoxicity on Saccharomyces cerevisiae cells – the role of mitochondria and YAP1 transcription factor

The present work aims to clarify the genotype differences of a model organism Saccharomyces cerevisiae in response to bee venom.The study evaluated various endpoints including cell survival, induction of physiologically active superoxide anions, mitotic gene conversion, mitotic crossing-over, reverse mutations, DNA double-strand breaks, and Ty1 retrotransposition. The role of the intact mitochondria and the YAP1 transcription factor was also evaluated.Our results indicate a genotype-specific response. The first experimental evidence has been provided that bee venom induces physiologically active superoxide anions and DNA double-strand breaks in S. cerevisiae. The lack of oxidative phosphorylation due to disrupted or missing mitochondrial DNA reduces but not diminishes the cytotoxicity of bee venom. The possible modes of action could be considered direct damage to membranes (cytotoxic effect) and indirect damage to DNA through oxidative stress (genotoxic effect). YAP1 transcription factor was not found to be directly involved in cell defense against bee venom treatment.

Transcriptional Profiling of Human Endothelial Cells Unveils PIEZO1 and Mechanosensitive Gene Regulation by Prooxidant and Inflammatory Inputs.

PIEZO1 is a mechanosensitive cation channel implicated in shear stress-mediated endothelial-dependent vasorelaxation. Since altered shear stress patterns induce a pro-inflammatory endothelial environment, we analyzed transcriptional profiles of human endothelial cells to determine the effect of altered shear stress patterns and subsequent prooxidant and inflammatory conditions on PIEZO1 and mechanosensitive-related genes (MRG). In silico analyses were validated in vitro by assessing PIEZO1 transcript levels in both the umbilical artery (HUAEC) and vein (HUVEC) endothelium. Transcriptional profiling showed that PIEZO1 and some MRG associated with the inflammatory response were upregulated in response to high (15 dyn/cm2) and extremely high shear stress (30 dyn/cm2) in HUVEC. Changes in PIEZO1 and inflammatory MRG were paralleled by p65 but not KLF or YAP1 transcription factors. Similarly, PIEZO1 transcript levels were upregulated by TNF-alpha (TNF-α) in diverse endothelial cell types, and pre-treatment with agents that prevent p65 translocation to the nucleus abolished PIEZO1 induction. ChIP-seq analysis revealed that p65 bonded to the PIEZO1 promoter region, an effect increased by the stimulation with TNF-α. Altogether this data showed that NF-kappa B activation via p65 signaling regulates PIEZO1 expression, providing a new molecular link for prooxidant and inflammatory responses and mechanosensitive pathways in the endothelium.

Abstract 4525: YAP1 in relapsed pulmonary high-grade neuroendocrine carcinomas (NEC) is associated with CDKN2A loss, intact RB1, EMT and therapeutic vulnerability to MEK1 and CDK4/6 inhibition

Abstract Neuroendocrine carcinomas (NECs) are clinically aggressive carcinomas commonly arising from the respiratory and gastrointestinal tracts, typically categorized as large-cell neuroendocrine carcinomas (LCNECs) or small cell carcinomas (most commonly small cell lung cancer (SCLC)). Clinically, pulmonary LCNECs (pLCNECs) mirror the course common to SCLC - initial response followed by rapid and insurmountable resistance to one-size-fits-all approaches. Recently, SCLC has been subdivided into four subtypes with unique vulnerabilities, three of which are defined by the transcription factors ASCL1, NEUROD1, and POU2F3, while a fourth group exhibits an inflamed signature. We hypothesize that pLCNEC may be similarly classified into molecularly distinct subsets with unique therapeutic vulnerabilities - a fundamental step toward personalized medicine. We applied our SCLC 1300 gene signature to pLCNEC patient tumors and, as in SCLC, found three distinct subtypes determined by differential expression of ASCL1, NEUROD1, and POU2F3, but with a unique fourth subtype marked by expression of the transcription factor YAP1. Unlike in treatment-naïve SCLC, where YAP1 is absent, YAP1 expression clearly defines pLCNEC as two, roughly equal subsets with the YAP1-low tumors encompassing tumors expressing the other three transcription factors. Conversely, YAP1-high pLCNEC is more mesenchymal and inflamed, and less neuroendocrine (NE), reminiscent of inflamed SCLC. Additionally, YAP1-high status is associated with smoking exposure (P<0.001, FC=81), high frequency of CDKN2A homozygous deletion and SMARCA4 mutations, as well as intact RB1. These features are distinct from SCLC, wherein transcriptional subtypes lack distinct genomic characteristics. Consistent with CDKN2A deletion, YAP1-high pLCNEC cell lines have increased sensitivity to MEK1 and CDK4/6 inhibition. We also demonstrate that RB1 loss downregulates YAP1 expression, which may account for the absence of YAP1 in treatment-naïve SCLC due to ubiquitous loss of RB1. In contrast to treatment-naïve SCLC, where our group and others have been unable to detect YAP1, single-cell RNAseq analysis of biopsies from patients with relapsed SCLC identified emerging YAP1-positive cancer cell populations, which are similarly associated with increased EMT, immune cell infiltration (CD8+ T-cells), and loss of NE gene expression. This suggests that the ability for cancer cells to acquire YAP1 expression and, perhaps, pLCNEC-like features, may be a resistance mechanism in relapsed SCLC, contributing to the abundant intratumoral heterogeneity and highlighting potential vulnerabilities to overcome resistance. In summary, YAP1 may be a predictive biomarker of intact RB1 and response to cellular and checkpoint immunotherapy and MEK1/CDK4/6 inhibition in pLCNEC and relapsed SCLC. Citation Format: C. Allison Stewart, Lixia Diao, Yuanxin Xi, Runsheng Wang, Kavya Ramkumar, B. Leticia Rodriguez, Benjamin B. Morris, Li Shen, Bingnan Zhang, Yan Yang, Azusa Tanimoto, Veronica Y. Novegil, Luisa M. Solis Soto, Pedro F. Simoes da Rocha, Natalie Vokes, Don L. Gibbons, Michael Frumovitz, Junya Fujimoto, Jing Wang, Bonnie Glisson, Lauren A. Byers, Carl M. Gay. YAP1 in relapsed pulmonary high-grade neuroendocrine carcinomas (NEC) is associated with CDKN2A loss, intact RB1, EMT and therapeutic vulnerability to MEK1 and CDK4/6 inhibition. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4525.

Role of AtYap1 in the reactive oxygen species regulation of lovastatin production in Aspergillus terreus

Lovastatin has great medical and economic importance, and its production in Aspergillus terreus is positively regulated at transcriptional level, by reactive oxygen species (ROS) generated during idiophase. To investigate the role of the transcription factor Yap1 in the regulation of lovastatin biosynthesis by ROS, an orthologue of yap1 was identified in A. terreus TUB F-514 and knocked down (silenced) by RNAi. Results confirmed that the selected knockdown strain (Siyap1) showed decreased yap1 expression in both culture systems (submerged and solid-state fermentation). Transformants showed higher sensitivity to oxidative stress. Interestingly, knockdown mutant showed higher ROS levels in idiophase and an important increase in lovastatin production in submerged and solid-state fermentations: 60 and 70% increase, respectively. Furthermore, sporulation also increased by 600%. This suggested that AtYap1 was functioning as a negative regulator of the biosynthetic genes, and that lack of AtYap1 in the mutants would be derepressing these genes and could explain increased production. However, we have shown that lovastatin production is proportional to ROS levels, so ROS increase in the mutants alone could also be the cause of production increase. In this work, when ROS levels were decreased with antioxidant, to the levels shown by the parental strain, the lovastatin production and kinetics were similar to the ones of the parental strain. This means that AtYap1 does not regulate lovastatin biosynthetic genes, and that production increase observed in the knockdown strain was an indirect effect caused by ROS increase. This conclusion is compared with studies on other secondary metabolites produced by other fungal species. KEY POINTS: • ROS regulates lovastatin biosynthesis at transcriptional level, in solid-state, and in submerged fermentations. • ATyap1 knockdown mutants showed important lovastatin production increases (60 and 70%) and higher ROS levels. • When ROS were decreased in the silenced mutant to the parental strain's level, lovastatin kinetics were identical to the parental strain's.

Manganese Stress Tolerance Depends on Yap1 and Stress-Activated MAP Kinases

Understanding which intracellular signaling pathways are activated by manganese stress is crucial to decipher how metal overload compromise cellular integrity. Here, we unveil a role for oxidative and cell wall stress signaling in the response to manganese stress in yeast. We find that the oxidative stress transcription factor Yap1 protects cells against manganese toxicity. Conversely, extracellular manganese addition causes a rapid decay in Yap1 protein levels. In addition, manganese stress activates the MAPKs Hog1 and Slt2 (Mpk1) and leads to an up-regulation of the Slt2 downstream transcription factor target Rlm1. Importantly, Yap1 and Slt2 are both required to protect cells from oxidative stress in mutants impaired in manganese detoxification. Under such circumstances, Slt2 activation is enhanced upon Yap1 depletion suggesting an interplay between different stress signaling nodes to optimize cellular stress responses and manganese tolerance.

The role of peroxiredoxins PrxA and PrxB in the antioxidant response, carbon utilization and development in Aspergillus nidulans

In addition to their role in the breakdown of H2O2, some peroxiredoxins (Prxs) have chaperone and H2O2 sensing functions. Acting as an H2O2 sensor, Prx Gpx3 transfers the oxidant signal to the transcription factor Yap1, involved in the antioxidant response in Saccharomyces cerevisiae. We have shown that Aspergillus nidulans Yap1 ortholog NapA is necessary for the antioxidant response, the utilization of arabinose, fructose and ethanol, and for proper development. To address the Prx roles in these processes, we generated and characterized mutants lacking peroxiredoxins PrxA, PrxB, PrxC, or TpxC. Our results show that the elimination of peroxiredoxins PrxC or TpxC does not produce any distinguishable phenotype. In contrast, the elimination of atypical 2-cysteine peroxiredoxins PrxA and PrxB produce different mutant phenotypes. ΔprxA, ΔnapA and ΔprxA ΔnapA mutants are equally sensitive to H2O2 and menadione, while PrxB is dispensable for this. However, the sensitivity of ΔprxA and ΔprxA ΔnapA mutants is increased by the lack of PrxB. Moreover, PrxB is required for arabinose and ethanol utilization and fruiting body cell wall pigmentation. PrxA expression is partially independent of NapA, and the replacement of peroxidatic cysteine 61 by serine (C61S) is enough to cause oxidative stress sensitivity and prevent NapA nuclear accumulation in response to H2O2, indicating its critical role in H2O2 sensing. Our results show that despite their high similarity, PrxA and PrxB play differential roles in Aspergillus nidulans antioxidant response, carbon utilization and development.

Pexophagy is critical for fungal development, stress response, and virulence in Alternaria alternata.

Alternaria alternata can resist high levels of reactive oxygen species (ROS). The protective roles of autophagy or autophagy‐mediated degradation of peroxisomes (termed pexophagy) against oxidative stress remain unclear. The present study, using transmission electron microscopy and fluorescence microscopy coupled with a GFP‐AaAtg8 proteolysis assay and an mCherry tagging assay with peroxisomal targeting tripeptides, demonstrated that hydrogen peroxide (H2O2) and nitrogen depletion induced autophagy and pexophagy. Experimental evidence showed that H2O2 triggered autophagy and the translocation of peroxisomes into the vacuoles. Mutational inactivation of the AaAtg8 gene in A. alternata led to autophagy impairment, resulting in the accumulation of peroxisomes, increased ROS sensitivity, and decreased virulence. Compared to the wild type, ΔAaAtg8 failed to detoxify ROS effectively, leading to ROS accumulation. Deleting AaAtg8 down‐regulated the expression of genes encoding an NADPH oxidase and a Yap1 transcription factor, both involved in ROS resistance. Deleting AaAtg8 affected the development of conidia and appressorium‐like structures. Deleting AaAtg8 also compromised the integrity of the cell wall. Reintroduction of a functional copy of AaAtg8 in the mutant completely restored all defective phenotypes. Although ΔAaAtg8 produced wild‐type toxin levels in axenic culture, the mutant induced a lower level of H2O2 and smaller necrotic lesions on citrus leaves. In addition to H2O2, nitrogen starvation triggered peroxisome turnover. We concluded that ΔAaAtg8 failed to degrade peroxisomes effectively, leading to the accumulation of peroxisomes and the reduction of the stress response. Autophagy‐mediated peroxisome turnover could increase cell adaptability and survival under oxidative stress and starvation conditions.

Abstract 992: MUC13 enhances colorectal cancer metastasis through molecular interaction with YAP1 transcription factor

Abstract Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the United States. About 90% of all cancer-related deaths are due to the development of metastatic sites in the body. 40-50% of colorectal cancer patients develop metastasis at some point during their fight with the disease. Understanding the mechanism of metastasis in colorectal cancer is vital. Metastasis is a multistep process; anchorage-independent survival after intravasation of cells from the primary tumor site is a crucial step. In our lab, we have previously demonstrated that MUC13 plays an essential role in the CRC progression by modulating anti-apoptotic pathways and survival proteins expression. Under anchorage-independent growth conditions, we have identified that MUC13, in correspondence with YAP1, is the key upregulated protein and is responsible for increased survival. Isogenic CRC cell lines SW480 (non-metastatic) and SW620 (metastatic) on low adherence growth conditions are applied in these studies. In addition, lentiviral transduced stable overexpression and knockdown cell lines were generated for MUC13 and YAP1 mechanistic studies. The overexpression of MUC13 in non-metastatic SW480 cells (low MUC13 expressing) increased anchorage-independent survival and enhanced tumorigenesis compared to SW480+Vector cells, contrary results were found upon MUC13 knockdown in SW620 cells (high MUC13 expressing). In vitro results were recapitulated in the in vivo mouse model system and human CRC tissues. In Proximity Ligation Assays (PLA), we found an increased nuclear localization of the survival complex YAP1/β-catenin in MUC13 overexpressing cells. In contrast, MUC13 knockdown resulted to the lower aboudance of survival complex in the nucleus. Immunoprecipitation validates the protein-protein interaction between MUC13 and YAP1. YAP1 knockdown in MUC13 overexpressing cells showed a decrease in survival, indicating the necessary functional complex formation between MUC13 and YAP1. MUC13 and YAP1 expression in human CRC tissue were highest at Stage II. However, YAP1 expression increased when MUC13 was observed in the nucleus. This supports the notion that MUC13 is critical in enhancing CRC metastasis through molecular interaction with YAP1. For the first time, this study demonstrates complex formation between MUC13 and YAP1 and defines their role in CRC progression and metastasis. Citation Format: Kyle Doxtater, Manish K. Tripathi, Radhika Sekhri, Sudhir Kotnala, Bilal Hafeez, Sheema Khan, Nadeem Zafar, Murali Yallapu, Meena Jaggi, Subhash Chauhan. MUC13 enhances colorectal cancer metastasis through molecular interaction with YAP1 transcription factor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 992.

368-OR: Activation of a Yap1-Dependent Mesenchymal Program Promotes ß-Cell Failure

A critical contributor to the pathogenesis of type 2 diabetes is the failure of insulin-secreting β-cells due to loss of cell number or identity. However, the signaling pathways that trigger these events remain unclear. While β-cells are of endodermal origin, recent studies show endocrine cells that coexpress the mesenchymal marker vimentin in the pancreas with diabetes, although the functional features of these cells are not known. Here, we identify an epithelial-to-mesenchymal (EMT) -like program that is activated in islets of mice and humans with type 2 diabetes. Characteristics of this EMT-like program include increased protein and mRNA abundance of the EMT transcription factor Snail and the mesenchymal adhesion molecule N-cadherin. Lineage-tracing techniques demonstrate that this mesenchymal program is initiated in β-cells that become multi-hormonal, a hallmark of dysfunctional endocrine cells. Interestingly, EMT-like cells minimally express Aldh1a3, a marker of dedifferentiated β-cells. To determine the mechanism driving EMT-like activation in β-cells, we tested stressors that phenocopy the effects of diabetes in islets. Treatment of islets with mitochondrial decouplers as well as oxidative stress increased expression of EMT markers. The transcription factor Yap1 is a known inducer of EMT. While Yap1 is expressed at minimal levels in β-cells, its abundance increased in db/db islets and in primary cells with impaired mitochondrial function. Importantly, chemical inhibition of Yap1 in db/db islets reversed activation of EMT-like factors. Yap1 inhibition also improved insulin secretion. Taken together, we found activation of an EMT-like program in the endocrine pancreas of mice and humans with type 2 diabetes. This program is dependent on increases in Yap1 due to oxidative stress and impaired mitochondrial function, and results in disrupted β-cell function. Disclosure W.Mckimpson: n/a. N.Hoque: None. J.Son: None. A.Y.Lee: None. D.Accili: Advisory Panel; Lilly Diabetes. Funding NIDDK (1K01DK121873)

Abstract P149: Novel compounds to probe Hippo kinase STK3 noncanonical function in prostate cancer

Abstract Prostate cancer (PC) is the second cause of cancer related deaths in men. Most therapies target the Androgen Receptor (AR) which can prolong survival, but resistance emerges and patients eventually succumb to the disease. This highlights the need for new PC molecular targets. Our group observed in PC patient datasets, the Serine/threoninekinase3 (STK3) gene is frequently amplified and correlates with worse outcomes. This is against the established dogma that STK3 is a tumor suppressor in the Hippo Tumor suppressor pathway that canonically regulates oncogenic transcription factor YAP1. Counter to this role, we report that in PC, STK3 has a pro-tumorigenic role. Our work shows that genetic and chemical inhibition of STK3 in PC cells in vitro significantly slowed proliferation and protasphere growth. The goal of this study was to identify and validate novel STK3 kinase inhibitors with increased potency and specificity. Approach. We screened large assay panels of kinase inhibitors (ChEMBL) for STK3 active molecules, which identified two independent scaffolds. For assessment of STK3 inhibition, we utilized a battery of in cell and biochemical assays including western blot, STK3-NanoBRET assays, radiometric enzymatic assays and a broad KINOMEscan scanMax panels to assess leads and analogs. For phenotypic studies, we utilized Hi-myc ventral prostate (HMVP2) cells to test STK3 inhibition in 3D spheroid growth and spheroid invasion of matrigel by live cell imaging. Pharmacokinetics (PK) and pharmacodynamics (PD) were performed for oral (PO) and intraperitoneal (IP) routes of our top STK3 inhibitor. A subcutaneous HMVP2 allograft model was used to test in vivo efficacy of STK3 inhibition by daily IP injections at 10 mg/kg and 30 mg/kg doses vs. vehicle. Tumor volumes were tracked by caliper measurements and at endpoint, we measured tumor, liver, lung, heart and spleen weights. Results. The two lead scaffolds and a number of analogs showed significant inhibition of STK3 by western blot analysis, in cell STK3-NanoBRET assay and recombinant STK3 radiometric assays. Phenotypic studies in HMVP2 spheres treated with analog STK3 inhibitors showed dose dependent decrease in proliferation and matrigel invasion. From our assays, compound SOB-5-47 emerged as the most potent. PK and PD studies showed that IP injections of SOB-5-47 led to short time in plasma, but pro-longed half-life in the organs such as liver, heart and notably prostate with a terminal half-life clearance of 8.3hrs. Daily injections of SOB-5-47 at 10 and 30 mgs/kg significantly delayed HMVP2 tumor kinetics compared to vehicle control. Tumor weights in SOB-5-47 groups were significantly lower vs control. Importantly, no differences in organ weights or total body weights were observed between groups. Conclusions. Here we report a noncanical role of STK3 in PC and the development of novel STK3 kinase inhibitors that can be used as tools to further examine the role of STK3. Additionally, we show that STK3 can be targeted in vivo to slow PC growth without significant unwanted deleterious effects. Citation Format: Amelia U. Schirmer, Lucy M. Driver, Megan T. Zhao, Carrow I. Wells, Xuan Yang, David H. Drewry, Ivan Spasojevic, Everardo Macias. Novel compounds to probe Hippo kinase STK3 noncanonical function in prostate cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P149.

The NADPH Oxidase A of Verticillium dahliae Is Essential for Pathogenicity, Normal Development, and Stress Tolerance, and It Interacts with Yap1 to Regulate Redox Homeostasis.

Maintenance of redox homeostasis is vital for aerobic organisms and particularly relevant to plant pathogens. A balance is required between their endogenous ROS production, which is important for their development and pathogenicity, and host-derived oxidative stress. Endogenous ROS in fungi are generated by membrane-bound NADPH oxidase (NOX) complexes and the mitochondrial respiratory chain, while transcription factor Yap1 is a major regulator of the antioxidant response. Here, we investigated the roles of NoxA and Yap1 in fundamental biological processes of the important plant pathogen Verticillium dahliae. Deletion of noxA impaired growth and morphogenesis, compromised formation of hyphopodia, diminished penetration ability and pathogenicity, increased sensitivity against antifungal agents, and dysregulated expression of antioxidant genes. On the other hand, deletion of yap1 resulted in defects in conidial and microsclerotia formation, increased sensitivity against oxidative stress, and down-regulated antioxidant genes. Localized accumulation of ROS was observed before conidial fusion and during the heterokaryon incompatibility reaction upon nonself fusion. The frequency of inviable fusions was not affected by the deletion of Yap1. Analysis of a double knockout mutant revealed an epistatic relationship between noxA and yap1. Our results collectively reveal instrumental roles of NoxA and ROS homeostasis in the biology of V. dahliae.

Improving the design of an oxidative stress sensing biosensor in yeast

ABSTRACTTranscription factor (TF)-based biosensors have proven useful for increasing biomanufacturing yields, large-scale functional screening, and in environmental monitoring. Most yeast TF-based biosensors are built from natural promoters, resulting in large DNA parts retaining considerable homology to the host genome, which can complicate biological engineering efforts. There is a need to explore smaller, synthetic biosensors to expand the options for regulating gene expression in yeast. Here, we present a systematic approach to improving the design of an existing oxidative stress sensing biosensor in Saccharomyces cerevisiae based on the Yap1 transcription factor. Starting from a synthetic core promoter, we optimized the activity of a Yap1-dependent promoter through rational modification of a minimalist Yap1 upstream activating sequence. Our novel promoter achieves dynamic ranges of activation surpassing those of the previously engineered Yap1-dependent promoter, while reducing it to only 171 base pairs. We demonstrate that coupling the promoter to a positive-feedback-regulated TF further improves the biosensor by increasing its dynamic range of activation and reducing its limit of detection. We have illustrated the robustness and transferability of the biosensor by reproducing its activity in an unconventional probiotic yeast strain, Saccharomyces boulardii. Our findings can provide guidance in the general process of TF-based biosensor design.

Impact of stress-response related transcription factor overexpression on lignocellulosic inhibitor tolerance of Saccharomyces cerevisiae environmental isolates.

Numerous transcription factor genes associated with stress response are upregulated in Saccharomyces cerevisiae grown in the presence of inhibitors that result from pretreatment processes to unlock simple sugars from biomass. To determine if overexpression of transcription factors could improve inhibitor tolerance in robust S. cerevisiae environmental isolates as has been demonstrated in S. cerevisiae haploid laboratory strains, transcription factors were overexpressed at three different expression levels in three S. cerevisiae environmental isolates. Overexpression of the YAP1 transcription factor in these isolates did not lead to increased growth rate or reduced lag in growth, and in some cases was detrimental, when grown in the presence of either lignocellulosic hydrolysates or furfural and 5-hydroxymethyl furfural individually. The expressed Yap1p localized correctly and the expression construct improved inhibitor tolerance of a laboratory strain as previously reported, indicating that lack of improvement in the environmental isolates was due to factors other than nonfunctional expression constructs or mis-folded protein. Additional stress-related transcription factors, MSN2, MSN4, HSF1, PDR1, and RPN4, were also overexpressed at three different expression levels and all failed to improve inhibitor tolerance. Transcription factor overexpression alone is unlikely to be a viable route toward increased inhibitor tolerance of robust environmental S. cerevisiae strains.

Telomere dysfunction activates YAP1 to drive tissue inflammation

Germline telomere maintenance defects are associated with an increased incidence of inflammatory diseases in humans, yet whether and how telomere dysfunction causes inflammation are not known. Here, we show that telomere dysfunction drives pATM/c-ABL-mediated activation of the YAP1 transcription factor, up-regulating the major pro-inflammatory factor, pro-IL-18. The colonic microbiome stimulates cytosolic receptors activating caspase-1 which cleaves pro-IL-18 into mature IL-18, leading to recruitment of interferon (IFN)-γ-secreting T cells and intestinal inflammation. Correspondingly, patients with germline telomere maintenance defects exhibit DNA damage (γH2AX) signaling together with elevated YAP1 and IL-18 expression. In mice with telomere dysfunction, telomerase reactivation in the intestinal epithelium or pharmacological inhibition of ATM, YAP1, or caspase-1 as well as antibiotic treatment, dramatically reduces IL-18 and intestinal inflammation. Thus, telomere dysfunction-induced activation of the ATM-YAP1-pro-IL-18 pathway in epithelium is a key instigator of tissue inflammation.

Identification of Sulfenylated Cysteines in Arabidopsis thaliana Proteins Using a Disulfide-Linked Peptide Reporter.

In proteins, hydrogen peroxide (H2O2) reacts with redox-sensitive cysteines to form cysteine sulfenic acid, also known as S-sulfenylation. These cysteine oxidation events can steer diverse cellular processes by altering protein interactions, trafficking, conformation, and function. Previously, we had identified S-sulfenylated proteins by using a tagged proteinaceous probe based on the yeast AP-1-like (Yap1) transcription factor that specifically reacts with sulfenic acids and traps them through a mixed disulfide bond. However, the identity of the S-sulfenylated amino acid residues within a protein remained enigmatic. By using the same transgenic YAP1C probe, we present here a technological advancement to identify in situ sulfenylated cysteine sites in Arabidopsis thaliana cells under control condition and oxidative stress. Briefly, the total extract of transgenic YAP1C A. thaliana cells was initially purified on IgG-Sepharose beads, followed by a tryptic digest. Then, the mixed disulfide-linked peptides were further enriched at the peptide level on an anti-YAP1C-derived peptide (C598SEIWDR) antibody. Subsequent mass spectrometry analysis with pLink 2 identified 1,745 YAP1C cross-linked peptides, indicating sulfenylated cysteines in over 1,000 proteins. Approximately 55% of these YAP1C-linked cysteines had previously been reported as redox-sensitive cysteines (S-sulfenylation, S-nitrosylation, and reversibly oxidized cysteines). The presented methodology provides a noninvasive approach to identify sulfenylated cysteines in any species that can be genetically modified.

The mitochondrial iron exporter genes MMT1 and MMT2 in yeast are transcriptionally regulated by Aft1 and Yap1

Budding yeast (Saccharomyces cerevisiae) responds to low cytosolic iron by up-regulating the expression of iron import genes; iron import can reflect iron transport into the cytosol or mitochondria. Mmt1 and Mmt2 are nuclearly encoded mitochondrial proteins that export iron from the mitochondria into the cytosol. Here we report that MMT1 and MMT2 expression is transcriptionally regulated by two pathways: the low-iron-sensing transcription factor Aft1 and the oxidant-sensing transcription factor Yap1. We determined that MMT1 and MMT2 expression is increased under low-iron conditions and decreased when mitochondrial iron import is increased through overexpression of the high-affinity mitochondrial iron importer Mrs3. Moreover, loss of iron-sulfur cluster synthesis induced expression of MMT1 and MMT2 We show that exposure to the oxidant H2O2 induced MMT1 expression but not MMT2 expression and identified the transcription factor Yap1 as being involved in oxidant-mediated MMT1 expression. We defined Aft1- and Yap1-dependent transcriptional sites in the MMT1 promoter that are necessary for low-iron- or oxidant-mediated MMT1 expression. We also found that the MMT2 promoter contains domains that are important for regulating its expression under low-iron conditions, including an upstream region that appears to partially repress expression under low-iron conditions. Our findings reveal that MMT1 and MMT2 are induced under low-iron conditions and that the low-iron regulator Aft1 is required for this induction. We further uncover an Aft1-binding site in the MMT1 promoter sufficient for inducing MMT1 transcription and identify an MMT2 promoter region required for low iron induction.

MAP2K1 is a potential therapeutic target in erlotinib resistant head and neck squamous cell carcinoma

Epidermal growth factor receptor (EGFR) targeted therapies have shown limited efficacy in head and neck squamous cell carcinoma (HNSCC) patients despite its overexpression. Identifying molecular mechanisms associated with acquired resistance to EGFR-TKIs such as erlotinib remains an unmet need and a therapeutic challenge. In this study, we employed an integrated multi-omics approach to delineate mechanisms associated with acquired resistance to erlotinib by carrying out whole exome sequencing, quantitative proteomic and phosphoproteomic profiling. We observed amplification of several genes including AXL kinase and transcription factor YAP1 resulting in protein overexpression. We also observed expression of constitutively active mutant MAP2K1 (p.K57E) in erlotinib resistant SCC-R cells. An integrated analysis of genomic, proteomic and phosphoproteomic data revealed alterations in MAPK pathway and its downstream targets in SCC-R cells. We demonstrate that erlotinib-resistant cells are sensitive to MAPK pathway inhibition. This study revealed multiple genetic, proteomic and phosphoproteomic alterations associated with erlotinib resistant SCC-R cells. Our data indicates that therapeutic targeting of MAPK pathway is an effective strategy for treating erlotinib-resistant HNSCC tumors.