YAP Expression Research Articles

The oncogenic role of NF1 in gallbladder cancer through regulation of YAP1 stability by direct interaction with YAP1

BackgroundGallbladder cancer (GBC) is the most prevalent and invasive biliary tract malignancy. As a GTPase-activating protein, Neurofibromin 1 (NF1) is a tumor suppressor that negatively regulates the RAS signaling pathway, and its abnormality leads to neurofibromatosis type 1 (NF-1) disease. However, the role of NF1 playing in GBC and the underlying molecular mechanism has not been defined yet.MethodsA combination of NOZ and EH-GB1 cell lines as well as nude mice, were utilized in this study. mRNA expression and protein levels of NF1 and YAP1 were evaluated by quantitative real-time PCR (qRT-PCR), western blot (WB), and immunohistochemistry (IHC). In vitro and in vivo assays were performed to explore the biological effects of NF1 in NOZ and EH-GB1 cells via siRNA or lv-shRNA mediated knockdown. Direct interaction between NF1 and YAP1 was detected by confocal microscopy and co-immunoprecipitation (Co-IP), and further confirmed by GST pull-down assay and isothermal titration calorimetry assay (ITC). The stability of proteins was measured by western blot (WB) in the presence of cycloheximide.ResultsThis study showed that a higher level of NF1 and YAP1 was found in GBC samples than in normal tissues and associated with worse prognoses. The NF1 knockdown impaired the proliferation and migration of NOZ in vivo and in vitro by downregulating YAP1 expression. Moreover, NF1 co-localized with YAP1 in NOZ and EH-GB1 cells, and the WW domains of YAP1 specifically recognized the PPQY motif of NF1. The structural modeling also indicated the hydrophobic interactions between YAP1 and NF1. On the other hand, YAP1 knockdown also impaired the proliferation of NOZ in vitro, phenocopying the effects of NF1 knockdown. Overexpression of YAP1 can partially rescue the impaired proliferation in NF1 stably knockdown cells. In mechanism, NF1 interacted with YAP1 and increased the stability of YAP1 by preventing ubiquitination.ConclusionsOur findings discovered a novel oncogenic function of NF1 by directly interacting with YAP1 protein and stabilizing YAP1 to protect it from proteasome degradation in NOZ cells. NF1 may serve as a potential therapeutic target in GBC.

Dihydroartemisinin inhibited interleukin-18 expression by decreasing YAP1 in hepatocellular carcinoma cells

BackgroundYes-associated protein 1 (YAP1) is highly expressed in liver cancer and has been used as an independent prognostic marker for hepatocellular carcinoma (HCC), while inhibition of YAP1 slows down the progression of HCC. Interleukin-18 (IL-18) also tends to be highly expressed in liver cancer. Previous research has proved that dihydroartemisinin (DHA) plays an important role in HCC treatment by reducing YAP1 expression. However, the relationship between YAP1 and IL-18 has not been reported in HCC, especially during DHA therapy. ObjectiveThe purpose of this study was to clarify the relationship between YAP1 and IL-18 in HCC cells, and to explicit the role of IL-18 in the treatment of HCC by DHA. Methods and resultsWe found that YAP1 and IL-18 were highly expressed in patients with hepatocellular carcinoma by bioinformatics analysis. Moreover, YAP1 was positively correlated with IL18 in liver cancer. YAP1 and IL18 correlated with immune cell infiltration, notably T cell exhaustion. YAP1 knockdown decreased IL-18 expression, while YAP1 overexpression increased the IL-18 expression in HCC cells. DHA reduced IL-18 expression through YAP1 in HCC cells. Further, DHA reduced the growth of Hepa1–6 cells subcutaneous xenograft tumors by inhibiting the expression of YAP1 and IL-18. However, DHA improved IL-18 in serum and adjacent tissues from DEN/TCPOBOP-induced liver tumor model in C57BL/6 mice. ConclusionYAP1 was positively correlated with IL-18 in HCC. DHA reduced the expression of IL-18 by inhibiting YAP1 and plays a role in the treatment of HCC. Our study suggested that IL-18 is a potential target for the treatment of HCC, and DHA is a promising drug for HCC therapy. Data availabilityThe dataset that supports the findings of this study is available from the corresponding author upon reasonable request.

Anti-Cancer Activity of Verteporfin in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a heterogenous malignancy that arises from the biliary epithelium and has a poor clinical prognosis. The Hippo/yes-associated protein (YAP) pathway has been reported to affect various aspects of tumorigenesis, with high expression of YAP1 being negatively associated with survival in CCA patients. Thus, we investigated the antitumoral effect of verteporfin, a YAP1 pathway inhibitor, in YAP1/AKT hydrodynamic tail vein injected murine models. We also used flow cytometry and single-cell RNA sequencing (scRNA-seq) to analyze the change in the immune cell profile and malignant cell stemness following verteporfin treatment. Our results demonstrated reduced liver weight and tumor formation in verteporfin-treated groups compared to that of a vehicle-treated group. Immune cell profiling through flow cytometry showed that relative to the vehicle, verteporfin induced a higher ratio of tumor-associated macrophage (TAM) M1/M2 and increased the percentage of activated CD8 T cell population (CD8+CD25+ and CD8+CD69+). scRNA-seq analysis showed significantly increased TAM M1 populations following verteporfin treatment and decreased proportions of stem-like cells within the malignant cell population. In summary, this study indicates that in CCA YAP/AKT murine models, verteporfin reduces tumorigenesis by polarizing anti-tumoral TAM and activating CD8 T cells and decreasing stem-like malignant cell proportions in the tumor microenvironment.

The interplay between IGF-1R signaling and Hippo-YAP in breast cancer stem cells

BackgroundBoth IGF-1R/PI3K/AKT/mTOR and Hippo pathways are crucial for breast cancer stem cells (BCSCs). However, their interplay remains unclear.MethodsFour triple negative breast cancer cell lines derived from CSC of two patient-derived xenografts (PDXs), AS-B145, AS-B145-1R, AS-B244, and AS-B244-1R, were used to elucidate the role of YAP in BCSCs. YAP silenced BCSCs were analyzed by cell proliferation, aldehyde dehydrogenase (ALDH) activity, mammosphere formation, and tumorigenesis. The effects of modulating IGF-1R and IGF-1 on YAP expression and localization were evaluated. The clinical correlation of YAP and IGF-1R signaling with the overall survival (OS) of 7830 breast cancer patients was analyzed by KM plotter.ResultsKnockdown of YAP abates the viability and stemness of BCSCs in vitro and tumorigenicity in vivo. Depletion of IGF-1R by shRNA or specific inhibitor decreases YAP expression. In contrast, IGF-1 addition upregulates YAP and enhances its nuclear localization. YAP overexpression increased the mRNA level of IGF-1, but not IGF-1R. Data mining of clinical breast cancer specimens revealed that basal-like breast cancer patients with higher level of IGF-1 and YAP exhibit significantly shorter OS.ConclusionsYAP contributes to stemness features of breast cancer in vitro and in vivo. The expression and localization of YAP was regulated by IGF-1R and YAP expression in turns upregulates IGF-1, but not IGF-1R. Clinically, higher level of YAP and IGF-1 significantly correlated with shorter OS in basal-like breast cancer. Taken together, these findings suggest the clinical relevance of interplay between YAP and IGF-1/IGF-1R pathway in sustaining the properties of BCSCs.DsuDGZeKddKHpMfgDukzC_Video

Shear and hydrostatic stress regulate fetal heart valve remodeling through YAP-mediated mechanotransduction.

Clinically serious congenital heart valve defects arise from improper growth and remodeling of endocardial cushions into leaflets. Genetic mutations have been extensively studied but explain less than 20% of cases. Mechanical forces generated by beating hearts drive valve development, but how these forces collectively determine valve growth and remodeling remains incompletely understood. Here, we decouple the influence of those forces on valve size and shape, and study the role of YAP pathway in determining the size and shape. The low oscillatory shear stress promotes YAP nuclear translocation in valvular endothelial cells (VEC), while the high unidirectional shear stress restricts YAP in cytoplasm. The hydrostatic compressive stress activated YAP in valvular interstitial cells (VIC), whereas the tensile stress deactivated YAP. YAP activation by small molecules promoted VIC proliferation and increased valve size. Whereas YAP inhibition enhanced the expression of cell-cell adhesions in VEC and affected valve shape. Finally, left atrial ligation was performed in chick embryonic hearts to manipulate the shear and hydrostatic stress in vivo. The restricted flow in the left ventricle induced a globular and hypoplastic left atrioventricular (AV) valves with an inhibited YAP expression. By contrast, the right AV valves with sustained YAP expression grew and elongated normally. This study establishes a simple yet elegant mechanobiological system by which transduction of local stresses regulates valve growth and remodeling. This system guides leaflets to grow into proper sizes and shapes with the ventricular development, without the need of a genetically prescribed timing mechanism.

Myoblast-derived exosomal Prrx2 attenuates osteoporosis via transcriptional regulation of lncRNA-MIR22HG to activate Hippo pathway

BackgroundSarcopenia and osteoporosis are common diseases that predominantly affect older individuals. The interaction between muscle and skeleton exerts pivotal roles in bone remodeling. This study aimed to explore the function of myoblast-derived exosomal Prrx2 in osteogenic differentiation and its potential mechanisms.MethodsExosomes were isolated from myogenic differentiated C2C12 cells. qRT-PCR and Western blotting were used to determine target molecule expression. Osteogenic differentiation of BMSCs was evaluated by Alizarin red staining, ALP activity and levels of OCN, OPN, RUNX2, and BMP2. Dual-luciferase reporter assay, RIP, and ChIP assays were performed to verify the interaction between molecules. The nuclear translocation of YAP1 was observed by immunofluorescence staining. In vivo osteoporotic model was established by ovariectomy in mice. Bone loss was examined using HE staining.ResultsPrrx2 expression was elevated in myogenic differentiated C2C12 cells and their exosomes. Myoblast-derived exosomal Prrx2 enhanced osteogenic differentiation of BMSCs. Delivering exosomal Prrx2 directly bond to MIR22HG promoter and promoted its transcription and expression. MIR22HG enhanced expression and nuclear translocation of YAP via sponging miR-128, thus facilitating BMSC osteogenic differentiation. Knockdown of exosomal Prrx2 suppressed osteogenic differentiation, which could be abolished by MIR22HG overexpression. Similarly, miR-128 inhibitor or YAP overexpression reversed the inhibitory effect of MIR22HG depletion or miR-128 mimics on osteogenic differentiation. Finally, myoblast-derived exosomal Prrx2 alleviated osteoporosis in mice via up-regulating MIR22HG and activating the Hippo pathway.ConclusionMyoblast-derived exosomal Prrx2 contributes to transcriptional activation of MIR22HG to activate YAP pathway via sponging miR-128, thereby facilitating osteogenic differentiation of BMSCs.

Anti-cancer activity of Chaga mushroom (Inonotus obliquus) against dog bladder cancer organoids.

Despite its disadvantages, chemotherapy is still commonly used for the treatment of bladder cancer (BC). Developing natural supplements that can target cancer stem cells (CSCs) which cause drug resistance and distant metastasis is necessary. Chaga mushrooms are popular to have several health-promoting and anti-cancer potentials. Organoid culture can recapitulate tumor heterogeneity, epithelial environment, and genetic and molecular imprints of the original tissues. In the previous study, we generated dog bladder cancer organoids (DBCO) as a novel experimental model of muscle-invasive BCO. Therefore, the present study aimed to examine the anti-tumor potentials of Chaga mushroom extract (Chaga) against DBCO. Four strains of DBCO were used in the present study. Treatment with Chaga inhibited the cell viability of DBCO in a concentration-dependent way. Treatment of DBCO with Chaga has significantly arrested its cell cycle and induced apoptosis. Expression of bladder CSC markers, CD44, C-MYC, SOX2, and YAP1, declined in the Chaga-treated DBCO. Also, Chaga inhibited the phosphorylation of ERK in DBCO. Expression of downstream signals of ERK, C-MYC, and Cyclins (Cyclin-A2, Cyclin-D1, Cyclin-E1, and CDK4) was also inhibited by Chaga in DBCO. Interestingly, the combinational treatment of DBCO with Chaga and anti-cancer drugs, vinblastine, mitoxantrone, or carboplatin, showed a potentiating activity. In vivo, Chaga administration decreased tumor growth and weight of DBCO-derived xenograft in mice with the induction of necrotic lesions. In conclusion, Chaga diminished the cell viability of DBCO by inhibiting proliferation-related signals and stemness conditions as well as by arresting the cell cycle. Collectively, these data suggest the value of Chaga as a promising natural supplement that could potentiate the effect of adjuvant chemotherapy, lower its adverse effects, and thus, limit the recurrence and metastasis of BC.

Expression Of Hippo-YAP Signaling Pathway in Gynecological Tumors

Ovarian cancer, breast cancer and cervical cancer are common female malignant tumors. YAP signal is widely activated in human malignant tumors. Hippo-YAP signal pathway affects the proliferation and development of tumors. However, the transmission mechanism of YAP signal in gynecological malignant tumors is still unclear. The function of YAP in the Hippo signal route is examined in this research, along with its functions upstream, middle, and downstream of the Hippo signal route. In addition, the mRNA expression data of YAP in ovarian serous cystadenocarcinoma and normal ovarian tissue is compared. The expression of YAP in breast invasive carcinoma (BRCA) is observed by IHC, and the YAP intensity of cytoplasm and nucleus is evaluated. Through the relationship between YAP and HPV, to further investigate the mechanism of YAP in cervical cancer, the distribution of YAP1 in cells adhering to the cervical surface was studied. These findings are critical in the treatment of YAP as a potential therapeutic target. Furthermore, understanding the role of YAP in female malignant tumors provide an insight in developing drugs that block the activity of YAP or inhibit the activation of YAP, they can play an anti-tumor role in vivo.

Whole-Section Landscape Analysis of Molecular Subtypes in Curatively Resected Small Cell Lung Cancer: Clinicopathologic Features and Prognostic Significance

Despite the recognition of various molecular subtypes in small cell lung cancer (SCLC), most information has been derived from tissue microarrays or biopsy samples. Using whole sections of curatively resected SCLCs, we aimed to elucidate the clinicopathologic relevance and prognostic significance of the molecular subtypes. Whole-section immunohistochemistry was conducted for 73 resected SCLC samples using antibodies representative of molecular subtypes: ASCL1 (SCLC-A), NEUROD1 (SCLC-N), POU2F3 (SCLC-P), and YAP1. Furthermore, multiplexed immunofluorescence was performed to evaluate the spatial relationship of YAP1 expression with other markers. The molecular subtype was correlated with clinical and histomorphologic features, and its prognostic role was explored in this cohort and validated in a previously published surgical cohort. Overall, the molecular subtypes were SCLC-A (54.8%), SCLC-N (31.5%), SCLC-P (6.8%), and SCLC-TN (triple negative, 6.8%). We found significant enrichment of SCLC-N (48.0%; P = .004) among combined SCLCs. Although a distinct subtype with high YAP1 expression was not found, YAP1 expression was reciprocal with ASCL1/NEUROD1 at the cellular level within tumors and was increased in areas with non–small cell–like morphology. Furthermore, the YAP1-positive SCLCs showed significantly increased recurrence at mediastinal lymph nodes (P = .047) and are an independent poor prognostic factor after surgery (adjusted hazard ratio, 2.87; 95% CI, 1.20-6.86; P = .017). The poor prognostic impact of YAP1 was also validated in the external surgical cohort. Our whole-section analysis in resected SCLCs reveals the highly heterogeneous nature of the molecular subtype and its clinicopathologic relevance. Although YAP1 is not a subtype delineator, YAP1 relates to the phenotypic plasticity of SCLC and may serve as a poor prognostic factor in resected SCLC.

FHL2 deficiency impairs follicular development and fertility by attenuating EGF/EGFR/YAP signaling in ovarian granulosa cells

Female subfertility is an increasing reproductive issue worldwide, which is partially related to abnormal ovarian follicular development. Granulosa cells (GCs), by providing the necessary physical support and microenvironment for follicular development, play critical roles in maintaining female fertility. We previously showed that ectopic expression of four and a half LIM domains 2 (FHL2) promoted ovarian granulosa cell tumor progression. However, its function in follicular development and fertility remains unknown. Here, we confirmed that FHL2 is highly expressed in human and mouse ovaries. FHL2 immunosignals were predominantly expressed in ovarian GCs. A Fhl2 knockout (KO) mouse model was generated to examine its roles in follicular development and fertility. Compared with wildtype, knockout of Fhl2 significantly decreased female litter size and offspring number. Furthermore, Fhl2 deficiency reduced ovarian size and impaired follicular development. RNA-sequencing analysis of GCs isolated from either KO or WT mice revealed that, Fhl2 deletion impaired multiple biological functions and signaling pathways, such as Ovarian Putative Early Atresia Granulosa Cell, ErbB, Hippo/YAP, etc. In vitro studies confirmed that FHL2 silencing suppressed GCs growth and EGF-induced GCs proliferation, while its overexpression promoted GC proliferation and decreased apoptosis. Mechanistic studies indicated that FHL2, via forming complexes with transcriptional factors AP-1 or NF-κB, regulated Egf and Egfr expression, respectively. Besides, FHL2 depletion decreased YAP1 expression, especially the active form of YAP1 (nuclear YAP1) in GCs of growing follicles. EGF, serving as an autocrine/paracrine factor, not only induced FHL2 expression and nuclear accumulation, but also stimulated YAP1 expression and activation. Collectively, our study suggests that FHL2 interacts with EGFR and Hippo/YAP signaling to regulate follicular development and maintain fertility. This study illuminates a novel mechanism for follicular development and a potential therapeutic target to address subfertility.

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.

Abstract 5825: TM4SF19 regulates oxidative stress-dependent YAP transcription in HNSCC

Abstract Head-and-neck squamous cell carcinoma (HNSCC), which arises from epithelial cells in mucosal surfaces of the upper aerodigestive tract, is the sixth leading cancer worldwide with 40-50% mortality rates. YAP is a transcriptional regulator that plays important roles in cancer cell proliferation and survival. HNSCC shows the highest levels of YAP gene expression among various types of human cancers. However, the molecular mechanisms explaining upregulation of YAP expression in HNSCC are poorly understood. Excessive consumption of tobacco and alcohol are classical risk factors of HNSCC. Tobacco and alcohol generates metabolites that increase reactive oxygen species (ROS) to abnormally high levels. It is not known whether YAP expression and function are interlinked with the oxidative stress response in HNSCC. Here, we aimed to elucidate the mechanism of YAP upregulation involved in the progression of HNSCC under conditions of oxidative stress. To identify candidates for YAP expression regulators in HNSCC, we analyzed whole-genome siRNA library screening data and TCGA genomics data. We performed loss-of-function and gain-of-function experiments to elucidate the molecular mechanism of TM4SF19, which is the top candidate for mediating transcriptional regulation of YAP in HNSCC. TM4SF19 is one of the transmembrane 4 L six family proteins that have topological similarities to tetraspanins, but little is known about its biological function. We found TM4SF19 controls the expression of YAP gene by regulating GABPβ1, a subunit of the GABP transcription factor complex, which is known to bind and activate YAP gene promoter. We observed that the depletion of TM4SF19 reduced GAPBPβ1, thereby decreasing the transcriptional activity of GABP complex. Importantly, we observed that TM4SF19 was dimerized in response to oxidative stress and consequently mediates the upregulation of YAP transcription by ROS elevation. We further show that TM4SF19 knockdown significantly reduces key characteristics of malignant cells, such as active proliferation and migration. Our results suggest that TM4SF19 is not only a novel transcriptional regulator of YAP but also a strong therapeutic target to suppress the oncogenic activity of YAP, and also provide new clue to understand HNSCC tumorigenesis involving YAP and oxidative stress. Citation Format: Eunbie Shin, Joon Kim. TM4SF19 regulates oxidative stress-dependent YAP transcription in HNSCC. [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 5825.

Abstract 1708: Sex-biased function of an oncogene, YAP1, regulates medulloblastoma stem cells and immune evasion

Abstract Sex disparity in both cancer incidence and response to therapeutics, especially immunotherapy, has become an important area of research in recent years. Brain tumors show obvious sex biases in incidence and therapy response. Medulloblastoma (MB) is the most common malignant pediatric brain tumor, and similar to other brain tumors, males have consistently higher incidence across all subgroups and at all ages. Moreover, for patients >3 years of age, females have significantly better outcomes than males. However, molecular drivers of such sex disparity in incidence and prognosis remain unclear.Here, we report a male-biased function of an oncogene, YAP1, in MB formation and immune suppression. Integrated multi-platform genomic, transcriptomic, proteomic, and in vivo transcriptional target analyses show that YAP1 promotes cancer stem cell maintenance through concurrent transcriptional activation of stemness genes, such as Sox2, and repression of differentiation regulators such as NeuroD1 and Zic1/2. YAP1 expression in MB cells also inhibits anti-tumor immune cell infiltration by directly regulating Csf1, Igf1, and Igfbp3 transcription and modulating IL6-JAK-STAT3, TNFR1, TGFb, and CCL5 Pathways. Notably, we found that YAP1 genetic deletion results in disproportionately higher survival of males than females in a spontaneous mouse model of infantile SHH MB, despite equivocal expression levels in both sexes. Consistent with our findings in mouse models, YAP1 downstream genes stratify MB patient survival in males but not females. Finally, we show that a brain-penetrant YAP1 inhibitor, verteporfin, extends survival and enhances immune infiltrates in vivo, mimicking the effects of Yap1 genetic deletion. In summary, this study demonstrates that an oncogene function may be sex-biased and underscores the need to incorporate sex as a variable in cancer mechanisms, tumor immunology, and treatment response studies. Citation Format: Nourhan Abdelfattah, Sivaraman Natarajan, Jose Maldonado, Han Nhat Tran, Hannah Borland, Rachael McMinimy, Shu-Hsia Chen, Fernando Camargo, James Olson, Joshy George, Kyuson Yun. Sex-biased function of an oncogene, YAP1, regulates medulloblastoma stem cells and immune evasion [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 1708.

Abstract 4995: Targeting PLK1 effectively suppresses growth of small cell lung cancer

Abstract Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor characterized by rapid disease progression and poor patient survival. Key transcription factors implicated as drivers of unique biological phenotypes of SCLC include ASCL1, NEUROD1, YAP1, and POU2F3. Strategies to exploit these phenotypes for innovative precision medicine approaches will be impactful. Using an unbiased agnostic preclinical drug screen to uncover therapeutic opportunities in SCLC, we identified a strong signal with PLK1 inhibitors (PLK1i) with low nanomolar IC50. We extended the in vitro findings by testing the efficacy of PLK1i in vivo using traditional xenograft of SCLC H526 cell line and patient derived xenografts (PDX). Volasertib achieved significant tumor growth inhibition relative to control in H526 xenografts. Also, onvansertib significantly inhibited growth of platinum-resistant and platinum-sensitive PDXs. The combination of PLK1i with standard chemotherapeutic agents identified promising synergy of the combination of onvansertib and paclitaxel. We further interrogated for predictive biomarkers of PLK1i sensitivity using gene expression profile comparing highly sensitive to less sensitive cell lines. High expression of C-MYC but not PLK1, TP53 or RB was associated with resistance to PLK1i. Conversely, while TP53 expression level did not correlate, TP53 gene mutation status (inactivating disruptive mutations) correlated with cell sensitivity to PLK1i. We queried the publicly available CCLE and the Cancer Therapeutics Response Portal to evaluate whether any of the SCLC subtypes have therapeutic vulnerability to PLK1i. In general, high expression of YAP1 in SCLC cell lines correlated with greater sensitivity to PLK1i. A YAP1 positive cell line, SW1271, with strong TP53 expression was particularly resistant to PLK1i. CRISPR knockout of YAP1 in this cell line enhanced SW1271 sensitivity to PLK1i suggesting that YAP1 expression as a marker of vulnerability to PLK1i could be context dependent especially when co-occurring with TP53 mutations. The mechanism of this interaction will be discussed. Citation Format: John C. Schmitz, Guojing Zhang, Andrey A. Ivanov, Abbe Pannucci, Maya Ridinger, Taofeek K. Owonikoko. Targeting PLK1 effectively suppresses growth of small cell lung cancer. [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 4995.

Abstract 1374: Small cell lung cancer predominantly expressing ASCL1 shows a distinct oxidative phenotype

Abstract Small cell lung cancer (SCLC) accounts for approximately 15% of lung cancers and has a poor prognosis due to a high proliferation rate and early metastasis. The current standard treatment, a combination of a platinum-based chemotherapy and etoposide, does not consider the underlying molecular profiles and, therefore, displays limited success. Tailoring treatment to the specific attributes of the 4 SCLC subtypes characterized by the predominant expression of either ASCL1, NeuroD1, POU2F3 or YAP1 (SCLC-A/N/P/Y) might improve patient outcomes. Here, we investigated the proteomic landscape of SCLC subsets with the aim to identify novel subtype-specific characteristics of diagnostic and therapeutic relevance. MS-based proteomics on 26 human SCLC cell lines was performed to decipher pathway-level differences between the subtypes. Mitochondria and lipid droplets were stained with Mitotracker Red CMXROS and Bodipy 493/503, respectively, and quantified by flow cytometry and confocal imaging. Differential responses to inhibitors of complex 1 (metformin, IACS-010759), the CPT1 inhibitor perhexiline, the glutaminolysis inhibitor BPTES and the aerobic glycolysis inhibitor UK5099 were tested using MTT-based cell viability assays. Pathway analyses based on proteomic data identified several metabolic pathways including “oxidative phosphorylation” (OXPHOS) to be significantly upregulated in SCLC-A compared to the other subtypes. Furthermore, multiple proteins essential for the electron transport chain (COX4I1, COX5B, NDUFA5) were highly expressed. Analysis of mitochondria showed a significantly higher number of mitochondria in cells (n=3) from the SCLC-A subtype, which also exhibited a more elongated shape. Lipid droplets were more abundant in the other, low OXPHOS subtypes (n=3). Perhexiline treatment confirmed higher OXPHOS activity solely in the SCLC-A subtype. In contrast, low OXPHOS cells were more sensitive to glutaminolysis inhibition, while no difference was seen when glycolysis was inhibited. Suggesting a differential response to clinically used OXPHOS inhibitors, SCLC cell lines with high and low oxidative activity were treated with metformin and IACS-010759 and response to treatment correlated positively with oxidative state. In this study, we demonstrate distinct differences in metabolic processes in the SCLC-A subtype compared to the other subtypes. Specifically, our data show upregulated oxidative phosphorylation as a potential therapeutic target. Understanding the variances in the metabolic phenotype between the molecular subtypes is integral to achieving precision medicine and advancing SCLC patient treatment. Citation Format: Karin Schelch, Anna Schwendenwein, Kristiina Boettiger, Dominik Kirchhofer, Christian Lang, Zsolt Megyesfalvi, Beata Szeitz, Konrad Hoetzenecker, Lukas Unger, Melinda Rezeli, Balazs Dome. Small cell lung cancer predominantly expressing ASCL1 shows a distinct oxidative phenotype [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 1374.

Abstract 6247: Lurbinectedin shows potent activity in all four molecular subtypes of small cell lung cancer (SCLC) and POU2F3 and SLFN11 are biomarkers for a better response

Abstract Background - SCLC is the most aggressive lung cancer type and with the worst prognosis. There are four molecular subtypes based on the high expression of distinct transcription factors and with different therapeutic vulnerabilities. However, all share transcriptional addiction as pathogenic mechanism. Lurbinectedin is a novel oncogenic transcription inhibitor, approved in the United States and other countries for the treatment of adult patients with metastatic SCLC with disease progression on or after platinum-based chemotherapy. The aim of this study was to analyze the activity of lurbinectedin in the different SCLC molecular subtypes and to investigate new biomarkers of response. Methods and Results - We have characterized a panel of 20 SCLC human cell lines based on the expression of ASCL1, NEUROD1, YAP1 and POU2F3, where lurbinectedin yielded a mean IC50 value of 6.52 nM, which is considerable greater than the activity exerted by topotecan, irinotecan, carboplatin, etoposide, olaparib, alisertib and navitoclax (IC50 100 µM-100 nM). Lurbinectedin potency is high in the four molecular subtypes, with IC50 values of 4.1, 14.9, 7.2, and 0.2 nM for SCLC-A, -N, -I and -P, respectively. However, high expression of POU2F3 correlated with better responses. In fact, mean IC50 was 0.28 nM for POU2F3high cells versus 12.1 nM for all POU2F3low cells (p=0.0443). Additionally, basal SLFN11 levels were evaluated, observing that SLFN11high cells responded better to lurbinectedin, with IC50 values of 1.1 nM versus 11.8 nM for SLFN11low (p=0.05). Likewise, lurbinectedin treatment induced greater antitumor activity in SLFN11high tumor-bearing mice (H526: T/C, 18% on day 11) than in SLFN11low tumor-bearing mice (H82: T/C, 65% on day 7) after intravenous treatment at 0.18 mg/kg (on days 0, 7 and 14). Finally, SLFN11 expression was evaluated by IHC in FFPE tumor samples from SCLC patients participating in a multicenter phase II clinical trial in advanced solid tumors (NCT02454972), which allowed lurbinectedin accelerated approval in this indication by FDA. Patients with higher expression of SLFN11 had a slightly better overall survival (OS at 6 months: 68.4% (<15%, N=20) vs. 98.7% (≥15%, N=20) p=0.0261)), especially in the refractory/resistant subgroup (OS at 6 months: 33.3% (<15%, N=9) vs. 100.0% (≥15%, N=6) p<0.0001). Conclusions - Lurbinectedin is highly effective in all molecular subtypes of SCLC in vitro and in vivo, with IC50 values at least two logs more potent than for other antitumoral agents and its activity is even greater in tumors with high POU2F3 expression and/or high SLFN11 expression. Citation Format: Marta Martínez Diez, Gema Santamaría Nuñez, María José Guillén, Daniel Rueda, Eva Maria Garrido-Martin, Pablo Avilés, Carmen Cuevas. Lurbinectedin shows potent activity in all four molecular subtypes of small cell lung cancer (SCLC) and POU2F3 and SLFN11 are biomarkers for a better response. [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 6247.

Abstract 1457: PDEF restricts prostate cancer cellular plasticity in part by modulating YAP1/TAZ-TEAD transcriptional network

Abstract Background: The Hippo TEAD-transcriptional regulators YAP1 and TAZ play essential role in cancer cell growth and metastasis. However, the function of YAP1 and TAZ in prostate cancer is not well characterized. We discovered that expression of PDEF is decreased during prostate cancer progression and re-expression of PDEF limits prostate cancer metastasis in part by promoting luminal epithelial phenotype, but mechanisms of PDEF action are not completely understood. In the present study, we evaluated the effects of the SPDEF on YAP1/TAZ levels and expression of YAP1/TAZ regulated genes in prostate cancer (PCa) cells. We also evaluated expression of SPDEF, YAP1, TAZ and YAPTAZZ-Tead regulated genes in clinical cohorts of prostate cancer patients. Material & Method: Prostate cancer (PC3 and DU145) cells were transfected with PDEF or respective vector control. shRNA was used to knock down SPDEF in AR positive and androgen responsive LNCaP cells. Protein levels were analyzed by western blotting. Gene expression was monitored by microarray analysis/RNA seq and confirmed by RTPCR assays. Cell migration and invasion were measured by scratch wound healing and by trans well migration though Matrigel assays respectively. We analyzed publicly available gene expression data in several prostate cancer cohorts. Results: We observed that the two well-characterized metastatic PCa cells (PC3 and DU145 cells) express both YAP1 and TAZ, but do not express SPDEF. Expression of PDEF in PC3 and DU145 cells resulted in an increased phospho-YAP1 and phospho-TAZ protein levels and inhibition of YAP1/TAZ target genes, as compared to the respective vector control, directly demonstrating that PDEF plays a critical role in modulating YAP1/TAZ-TEAD transcriptional activity, and by extension in the regulation of the Hippo pathway. Analysis of publicly available PCa data sets revealed a significant increase in TAZ mRNA levels in prostate cancer tissues as compared to normal prostate tissues (p=4.95E-08). Furthermore, we also found a significant gradual increase in TAZ mRNA expression and concomitant decrease in YAP1 mRNA during disease progression and metastasis and in neuroendocrine PCa. Conclusions: Taken together these data suggest that SPDEF limits prostate cancer metastasis in part by targeting YAP1/TAZ driven transcriptional output. Citation Format: Hari K. Koul, Praveen K. Jaiswal, Suman Mohajan, Mousa Vatanmakanian, Fengtian Wang, Sweaty Koul. PDEF restricts prostate cancer cellular plasticity in part by modulating YAP1/TAZ-TEAD transcriptional network [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 1457.

Abstract 6290: Characterizing antitumor responses to EXS-74539, a novel, reversible LSD1 inhibitor with potential in small-cell lung cancer

Abstract Background: Lysine demethylase 1 (LSD1) is a potentially important target in oncology due to its role in demethylating histones and suppressing expression of genes required for cellular differentiation. In small-cell lung cancer (SCLC), high LSD1 expression is associated with suppressed differentiation pathways. Although selective LSD1 compounds are in clinical development, understanding of the molecular markers of tumor sensitivity to inhibition of LSD1 inhibition is incomplete. Methods: We report on the pharmacological characterization of EXS-74539, a potent, selective and reversible LSD1 inhibitor. We assessed EXS-74539 efficacy in in vitro and in vivo SCLC cell line models, encompassing the four molecular subtypes of SCLC tumor cells previously proposed (Rudin et al, 2019), based on differential expression of key transcription regulators, ASCL1, NEUROD1, POU2F3 and YAP1. Results: In vitro, EXS-74539 demonstrated similar anti-proliferative activity against both neuroendocrine (NCI-H1417 and NCI-H69, ASCL1 sub-type) and non-neuroendocrine (NCI-H526: POU2F3) SCLC cell lines, with IC50 values of 28, 12 and 11 nM, respectively. In vitro exposure of these cell lines to EXS-74539 led to concentration-dependent alterations in differentiation-associated genes. However, in vivo, significant differences in antitumor growth response upon LSD1 inhibition were observed between the cell lines; NCI-H69 tumor growth was unaffected following treatment with EXS-74539 and an irreversible LSD1 inhibitor at dose regimens which were efficacious in the NCI-H1417 and NCI-H5126 models. We describe in vitro and in vivo gene expression correlations associated with LSD1 sensitivity. Conclusions: The reversible LSD1 inhibitor, EXS-74539, is a potent inhibitor of SCLC cell line proliferation and shows in vivo efficacy. Characterizing SCLC cell line sensitivity in vitro and in vivo identified gene signatures that may have use as markers of sensitivity, which we are currently characterizing and validating in human SCLC patient samples. Rudin et al., Nat Rev Cancer, 2019 Citation Format: Andrew Payne, Manisha Naik, Simon Richards, Victor Sebastian Perez, Maria Dominguez. Characterizing antitumor responses to EXS-74539, a novel, reversible LSD1 inhibitor with potential in small-cell lung cancer [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 6290.

Abstract 1612: Therapeutic co-targeting YAP1 and TAZ using antisense oligos (ASOs) suppress gastric cancer progression and peritoneal metastases

Abstract Gastric adenocarcinoma (GAC) is one of the most common malignancies worldwide and it is the fourth leading cause of cancer-related death. Peritoneal carcinomatosis (PC; malignant ascites or implants) in GAC patients is common and poses a challenge with short survival and lack of effective therapeutics. We and others have demonstrated that deregulation of the Hippo signaling pathway particularly with upregulation of its coactivators-YAP1 or TAZ drive cancer progression and metastases in gastroesophageal cancers suggesting YAP1 and TAZ are potential drug targets in solid tumors. However, discovery of effective drugs to target YAP1 or TAZ remains challenging due to the nuclear localization and lack of inhibitory pocket for YAP1 or TAZ. In this study, using scRNAseq and immunofluorescent staining, we observed that both YAP1, TAZ and their transcriptional factors-Tead1, Tead2, Tead3 and Tead4 are highly expressed in PC tumor cells and high expression of these proteins were associated with poorer prognosis. Further, we note that recently developed YAP or TAZ antisense oligonucleotides (ASO) can effectively and specifically suppress YAP or TAZ expression and transcription accompanied by decreased tumor cell invasion and tumor sphere formation. Further, we observed that YAP1 can interact with TAZ and both bind TEAD1,2,3 but not Tead4 transcriptional factors, while YAP1 ASO or TAZ ASO can decrease the interactions between YAP1 or TAZ with TEAD1,2,3 in GAC cells. Interestingly, inhibition of YAP1 or TAZ alone using the ASOs can complementarily increase the other at the protein and mRNA levels. Further, we revealed that YAP1 KO patient derived tumor cells (GA0518) are more sensitive to TAZ ASO than control cells and simultaneously inhibition of YAP1 and TAZ by ASOs reduces both YAP1and TAZ proteins and mRNA levels with significant decrease in cell proliferation and invasive capacity of YAP1 high tumor cells. Most importantly, co-targeting YAP and TAZ by the ASOs significantly attenuated progression and PC in the PDX model and sensitized to anti-PD1 immunotherapy in the KP-Luc syngeneic model. Taken together, our studies open a new avenue for developing novel therapeutic strategy by co-targeting both YAP1 and TAZ using the ASOs against GAC with PC. Keywords: Gastric Adenocarcinoma, YAP1, TAZ, Hippo pathway, Antisense Oligonucleotides (ASOs), Peritoneal Metastases, Targeted Therapy Citation Format: Jingjing Wu, Ailing Scott, Yan Xu, Yuan Li, Yibo Fan, Ruiping Wang, Xiaodan Yao, Katsuhiro Yoshimura, Melissa Pool Pizzi, Kohei Yamashita, Shan Shao, Christopher Vellano, Linghua Wang, Alexey Revenko, Eric Dolinski, Jaffer A. Ajani, Shumei Song. Therapeutic co-targeting YAP1 and TAZ using antisense oligos (ASOs) suppress gastric cancer progression and peritoneal metastases [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 1612.

Abstract 214: Patient-specific mapping of oncogenic structural changes in pediatric osteosarcoma

Abstract We have leveraged long-read and spatial sequencing modalities to identify patient-specific oncogenic alterations for treatment of pediatric osteosarcomas. Osteosarcoma (OS) is the most common primary bone malignancy in children and young adults, with over 500 cases identified each year in the United States. OS is archetypal of cancers driven by aneuploidy and structural rearrangement rather than point mutations. These structural variants (SVs) can alter gene expression stoichiometrically, but the functional consequences of many SVs stem from their effect on multidimensional genome organization. SVs can enable enhancer hijacking, alter boundaries of topologically associated domains (TADs), and move gene loci to different regulatory compartments affecting transcription. SVs can also form extrachromosomal DNA amplicons (ecDNA), which can be megabases in size and can incorporate both genes and regulatory elements to result in substantial increases in gene transcription and intratumor heterogeneity. Complex SVs may have profound implications for prognosis and treatment in OS and other cancers. By incorporating long-read optical genome mapping (OGM) and chromatin conformation capture sequencing (HiC), our goal is to detect complex tumor-specific SVs that will inform and refine pediatric OS subtypes, revealing new tumor-specific chemotherapy treatment options. We have generated a unique panel of 11 OS patient-derived xenograft (PDX) cell lines which have been previously characterized regarding response to targeted therapies using whole genome (WGS), RNA (RNA-seq), and chromatin accessibility (ATAC-seq) sequencing. To understand the complex genomic reorganization that occurs in OS, we have integrated OGM and WGS data for six cell lines, and OGM, WGS, and HiC data for two. SVs were detected using OGM, HiC, and WGS modalities, and we observed 59-61% concordance between SVs called using OGM and HiC, versus 22-45% when comparing with WGS calls. PDX lines assayed using OGM exhibit between 5,135 and 7,514 SVs each, and translocation numbers ranged from 212 to 1,422. These lines exhibit between dozens of potential ecDNA amplicons, including one amplicon which may explain that line’s specific and exceptionally high expression of YAP1 and BIRC2 transcripts. HiC interaction maps for two PDX lines reveal genomic compartments and TAD boundaries in detail, with 3,713 unique TAD boundaries identified between them using 50kb genomic bins. We observe that 60% of these boundaries are consistent between these lines, and 27% exhibit differences that suggest boundary loss or gain. These changing boundaries are associated with numerous SVs and/or copy number discrepancies, and with genomic regions containing genes with dysregulated RNA transcript levels. Incorporating OGM and HiC modalities adds substantially to our ability to detect oncogenic SVs and the genes they affect. Citation Format: Andrew S. Clugston, Megan Ostrowski, Coco Wu, Leanne Sayles, Stanley G. Leung, Vijay Ramani, E. Alejandro Sweet-Cordero, Marcus R. Breese. Patient-specific mapping of oncogenic structural changes in pediatric osteosarcoma [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 214.