Ets Family Research Articles

Circ_0022340 promotes colorectal cancer progression via HNRNPC/EBF1/SYT7 or miR-382-5p/ELK1 axis

Circular RNAs (circRNAs) are characterized as a class of new noncoding RNAs and function in tumorigenesis of colorectal cancer (CRC). In our study, the molecule mechanism of circ_0022340 in CRC was investigated. For this aim, quantitative real-time polymerase chain reaction (RT-qPCR) was used to test gene expression in CRC cells. Cell function assays including 5-ethynyl-20-deoxyuridine (EdU), colony formation and transwell investigated the proliferation and migration capacity in CRC cells. Luciferase reporter and RNA immunoprecipitation (RIP)assays determined the interaction between circRNA, miRNA and mRNA. Western blot was used to test protein expression. An immunohistochemistry assay was used to assess the tumor growth in vivo. Results showed that Circ_0022340 was highly expressed in CRC cells. Circ_0022340 was formed from exon 5 to 6 of the synaptotagmin 7 (SYT7). Silencing of circ_0022340 suppressed CRC cell proliferation and migration. Functionally, circ_0022340 recruited heterogeneous nuclear ribonucleoprotein C (HNRNPC) to stabilize EBF1 mRNA and thereby activated SYT7. Moreover, circ_0022340 targeted miR-382-5p to up-regulate ETS transcription factor ELK1 (ELK1). It is concluded that Circ_0022340 promoted colorectal cancer progression via recruiting HNRNPC to stabilize EBF1 mRNA and thereby activated SYT7 or miR-382-5p/ELK1 axis, which might provide a novel target for CRC treatment.

ELF4 is a critical component of a miRNA-transcription factor network and is a bridge regulator of glioblastoma receptor signaling and lipid dynamics.

The loss of neurogenic tumor suppressor microRNAs miR-124, miR-128, and miR-137 is associated with glioblastoma's undifferentiated state. Most of their impact comes via the repression of a network of oncogenic transcription factors. We conducted a high-throughput functional siRNA screen in glioblastoma cells and identify E74 like ETS transcription factor 4 (ELF4) as the leading contributor to oncogenic phenotypes. In vitro and in vivo assays were used to assess ELF4 impact on cancer phenotypes. We characterized ELF4's mechanism of action via genomic and lipidomic analyses. AMAPK reporter assay verified ELF4's impact on MAPK signaling, and qRT-PCR and western blotting were used to corroborate ELF4 regulatory role on most relevant target genes. ELF4 knockdown resulted in significant proliferation delay and apoptosis in GBM cells and long-term growth delay and morphological changes in glioma stem cells (GSCs). Transcriptomic analyses revealed that ELF4 controls two interlinked pathways: 1)Receptor tyrosine kinase signaling and 2)Lipid dynamics. ELF4 modulation directly affected receptor tyrosine kinase (RTK) signaling, as mitogen-activated protein kinase (MAPK) activity was dependent upon ELF4 levels. Furthermore, shotgun lipidomics revealed that ELF4 depletion disrupted several phospholipid classes, highlighting ELF4's importance in lipid homeostasis. We found that ELF4 is critical for the GBM cell identity by controlling genes of two dependent pathways: RTK signaling (SRC, PTK2B, and TNK2) and lipid dynamics (LRP1, APOE, ABCA7, PLA2G6, and PITPNM2). Our data suggest that targeting these two pathways simultaneously may be therapeutically beneficial to GBM patients.

Elk1/miR-462-731 Feedback Loop Regulates Macrophages Polarization and Phagocytosis in Grass Carp (Ctenopharyngodon idella).

MicroRNA clusters are microRNAs (miRNAs) that are distributed in close proximity on chromosomes. In this study, we report a miRNA cluster identified from grass carp (Ctenopharyngodon idella), miR-462-731, which plays a positive role in host antibacterial immunity. The expression of miR-462-731 was disrupted after infection by Aeromonas hydrophila. Transcription factor ETS transcription factor ELK1 was identified to bind to the promoter of the miR-462-731 cluster and suppress its expression. In addition, miR-731 negatively regulates the expression of elk1, forms an elk1/miR-462-731 double negative feedback loop. In addition, we found that miR-731 directly targets ezrin a (ezra), participates in inducing PI3K/AKT signaling in macrophage, to induce macrophage polarization to the M1 phenotype with stronger phagocytosis. Our results demonstrate a novel elk1/miR-462-731 feedback loop. The data deepen our understanding of the relationship between macrophage polarization and phagocytosis in teleost fish.

Signaling Pathway of Taurine-Induced Upregulation of TXNIP

Taurine, a sulfur-containing β-amino acid, is present at high concentrations in mammalian tissues and plays an important role in several essential biological processes. However, the genetic mechanisms involved in these physiological processes associated with taurine remain unclear. In this study, we investigated the regulatory mechanism underlying the taurine-induced transcriptional enhancement of the thioredoxin-interacting protein (TXNIP). The results showed that taurine significantly increased the luciferase activity of the human TXNIP promoter. Further, deletion analysis of the TXNIP promoter showed that taurine induced luciferase activity only in the TXNIP promoter region (+200 to +218). Furthermore, by employing a bioinformatic analysis using the TRANSFAC database, we focused on Tst-1 and Ets-1 as candidates involved in taurine-induced transcription and found that the mutation in the Ets-1 sequence did not enhance transcriptional activity by taurine. Additionally, chromatin immunoprecipitation assays indicated that the binding of Ets-1 to the TXNIP promoter region was enhanced by taurine. Taurine also increased the levels of phosphorylated Ets-1, indicating activation of Ets-1 pathway by taurine. Moreover, an ERK cascade inhibitor significantly suppressed the taurine-induced increase in TXNIP mRNA levels and transcriptional enhancement of TXNIP. These results suggest that taurine enhances TXNIP expression by activating transcription factor Ets-1 via the ERK cascade.

Optimization of synthetic molecular reporters for a mesenchymal glioblastoma transcriptional program by integer programing.

A recent approach to perform genetic tracing of complex biological problems involves the generation of synthetic deoxyribonucleic acid (DNA) probes that specifically mark cells with a phenotype of interest. These synthetic locus control regions (sLCRs), in turn, drive the expression of a reporter gene, such as fluorescent protein. To build functional and specific sLCRs, it is critical to accurately select multiple bona fide cis-regulatory elements from the target cell phenotype cistrome. This selection occurs by maximizing the number and diversity of transcription factors (TFs) within the sLCR, yet the size of the final sLCR should remain limited. In this work, we discuss how optimization, in particular integer programing, can be used to systematically address the construction of a specific sLCR and optimize pre-defined properties of the sLCR. Our presented instance of a linear optimization problem maximizes the activation potential of the sLCR such that its size is limited to a pre-defined length and a minimum number of all TFs deemed sufficiently characteristic for the phenotype of interest is covered. We generated an sLCR to trace the mesenchymal glioblastoma program in patients by solving our corresponding linear program with the software optimizer Gurobi. Considering the binding strength of transcription factor binding sites (TFBSs) with their TFs as a proxy for activation potential, the optimized sLCR scores similarly to an sLCR experimentally validated in vivo, and is smaller in size while having the same coverage of TFBSs. We provide a Python implementation of the presented framework in the Supplementary Material with which an optimal selection of cis-regulatory elements can be calculated once the target set of TFs and their binding strength with their TFBSs is known. Supplementary data are available at Bioinformatics online.

HDACs regulate the differentiation of endothelial cells from human iPSCs.

Human induced pluripotent stem cells (hiPSCs) possess the potential to differentiate toward vascular cells including endothelial cells (ECs), pericytes, and smooth muscle cells. Epigenetic mechanisms including DNA methylation and histone modification play a crucial role in regulating lineage differentiation and specification. Herein, we utilized a three-stage protocol to induce differentiation of mesoderm, vascular progenitors, and ECs from hiPSCs and investigated the regulatory effects of histone acetylation on the differentiation processes. We found that the expression of several histone deacetylases (HDACs), including HDAC1, HDAC5, and HDAC7, were greatly upregulated at the second stage and downregulated at the third stage. Interestingly, although HDAC1 remained in the nucleus during the EC differentiation, HDAC5 and HDAC7 displayed cytosol/nuclear translocation during the differentiation process. Inhibition of HDACs with sodium butyrate (NaBt) or BML210 could hinder the differentiation of vascular progenitors at the second stage and facilitate EC induction at the third stage. Further investigation revealed that HDAC may modulate the stepwise EC differentiation via regulating the expression of endothelial transcription factors ERG, ETS1, and MEF2C. Opposite to the expression of EC markers, the smooth muscle/pericyte marker ACTA2 was upregulated at the second stage and downregulated at the third stage by NaBt. The stage-specific regulation of ACTA2 by HDAC inhibition was likely through regulating the expression of TGFβ2 and PDGFB. This study suggests that HDACs play different roles at different stages of EC induction by promoting the commitment of vascular progenitors and impeding the later stage differentiation of ECs.

Transcription Factor RUNX3 Mediates Plasticity of ThGM Cells Toward Th1 Phenotype.

GM-CSF-producing T helper (Th) cells play a crucial role in the pathogenesis of autoimmune diseases such as multiple sclerosis (MS). Recent studies have identified a distinct population of GM-CSF-producing Th cells, named ThGM cells, that also express cytokines TNF, IL-2, and IL-3, but lack expression of master transcription factors (TF) and signature cytokines of commonly recognized Th cell lineages. ThGM cells are highly encephalitogenic in a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Similar to Th17 cells, in response to IL-12, ThGM cells upregulate expression of T-bet and IFN-γ and switch their phenotype to Th1. Here we show that in addition to T-bet, TF RUNX3 also contributes to the Th1 switch of ThGM cells. T-bet-deficient ThGM cells in the CNS of mice with EAE had low expression of RUNX3, and knockdown of RUNX3 expression in ThGM cells abrogated the Th1-inducing effect of IL-12. Comparison of ThGM and Th1 cell transcriptomes showed that ThGM cells expressed a set of TFs known to inhibit the development of other Th lineages. Lack of expression of lineage-specific cytokines and TFs by ThGM cells, together with expression of TFs that inhibit the development of other Th lineages, suggests that ThGM cells are a non-polarized subset of Th cells with lineage characteristics.

Integrative genome‐wide chromatin accessibility and transcriptome profiling of diffuse large B‐cell lymphoma

Integrative genome‐wide chromatin accessibility and transcriptome profiling of diffuse large B‐cell lymphoma

Ets1 mediates sorafenib resistance by regulating mitochondrial ROS pathway in hepatocellular carcinoma

The incidence and mortality of hepatocellular carcinoma (HCC) are on a rise in the Western countries including US, attributed mostly to late detection. Sorafenib has been the first-line FDA-approved drug for advanced unresectable HCC for almost a decade, but with limited efficacy due to the development of resistance. More recently, several other multi-kinase inhibitors (lenvatinib, cabozantinib, regorafenib), human monoclonal antibody (ramucirumab), and immune checkpoint inhibitors (nivolumab, pembrolizumab) have been approved as systemic therapies. Despite this, the median survival of patients is not significantly increased. Understanding of the molecular mechanism(s) that govern HCC resistance is critically needed to increase efficacy of current drugs and to develop more efficacious ones in the future. Our studies with sorafenib-resistant (soraR) HCC cells using transcription factor RT2 Profiler PCR Arrays revealed an increase in E26 transformation–specific-1 (Ets-1) transcription factor in all soraR cells. HCC TMA studies showed an increase in Ets-1 expression in advanced HCC compared to the normal livers. Overexpression or knocking down Ets-1 modulated sorafenib resistance-related epithelial–mesenchymal transition (EMT), migration, and cell survival. In addition, the soraR cells showed a significant reduction of mitochondrial damage and mitochondrial reactive oxygen species (mROS) generation, which were antagonized by knocking down Ets-1 expression. More in-depth analysis identified GPX-2 as a downstream mediator of Ets-1-induced sorafenib resistance, which was down-regulated by Ets-1 knockdown while other antioxidant pathway genes were not affected. Interestingly, knocking down GPX2 expression significantly increased sorafenib sensitivity in the soraR cells. Our studies indicate the activation of a novel Ets-1–GPX2 signaling axis in soraR cells, targeting which might successfully antagonize resistance and increase efficacy.

Crif1 is Required for Proper Mesenchymal to Epithelial Transition

The epithelial to mesenchymal and the mesenchymal to epithelial transitions (EMT and MET) are fundamental developmental processes required for shaping the embryo but are also hijacked by cancer cells during metastasis. The regulation of EMT is very well studied, and major regulators have been identified. A similar understanding of the regulation of MET is needed. Recently we and others have described essential factors for the initiation and progression of MET; among them is the ETS transcription factor Elf3. Recent reports showed that Crif1 is critical for the function of Elf3, yet the influence on MET has not been reported. Here, we studied the involvement of Crif1 in MET using a loss of function approach in NMuMG cells. We found that the depletion of Crif1 resulted in an impaired MET. We have also noticed that Cdh1 mRNA and protein expression was not affected; instead, E-cadherin, the protein product of Cdh1, was localized to the cytoplasm. These results are in agreement with our previous findings following the depletion of Elf3. In conclusion, Crif1 was essential for the mesenchymal to epithelial transition, and it may exert its function in cooperation with Elf3

De novo Transcriptome Analysis of Drought-Adapted Cluster Bean (Cultivar RGC-1025) Reveals the Wax Regulatory Genes Involved in Drought Resistance.

Cluster bean (Cyamopsis tetragonoloba L.) is one of the multipurpose underexplored crops grown as green vegetable and for gum production in dryland areas. Cluster bean is known as relatively tolerant to drought and salinity stress. To elucidate the molecular mechanisms involved in the drought tolerance of cluster bean cultivar RGC-1025, RNA sequencing (RNA-seq) of the drought-stressed and control samples was performed. De novo assembly of the reads resulted in 66,838 transcripts involving 203 pathways. Among these transcripts, differentially expressed gene (DEG) analysis resulted in some of the drought-responsive genes expressing alpha dioxygenase 2, low temperature-induced 65 kDa protein (LDI65), putative vacuolar amino acid transporter, and late embryogenesis abundant protein (LEA 3). The analysis also reported drought-responsive transcription factors (TFs), such as NAC, WRKY, GRAS, and MYB families. The relative expression of genes by qRT-PCR revealed consistency with the DEG analysis. Key genes involved in the wax biosynthesis pathway were mapped using the DEG data analysis. These results were positively correlated with epicuticular wax content and the wax depositions on the leaf surfaces, as evidenced by scanning electron microscope (SEM) image analysis. Further, these findings support the fact that enhanced wax deposits on the leaf surface had played a crucial role in combating the drought stress in cluster beans under drought stress conditions. In addition, this study provided a set of unknown genes and TFs that could be a source of engineering tolerance against drought stress in cluster beans.

P417: A SINGLE AMINO ACID AT THE PNT DOMAIN OF ERG MEDIATES ITS LEUKEMOGENIC ACTIVITY THROUGH INTERACTION WITH THE NCOR-HDAC3 CO-REPRESSOR COMPLEX.

Background: The ETS transcription factor ERG is essential for the maintenance of hematopoietic stem cells. However, it has also been implicated as an oncogene in the development of acute leukemia. Our studies and those of others have demonstrated that ERG directly contributes to the initiation and maintenance of lymphoid and myeloid acute leukemia subtypes. Nevertheless, ERG co-factors critically involved in leukemogenesis remain largely uncharacterized. Aims: Here we report a critical role for the conserved amino-acid proline at position 199, at the 3’ end of the PNT domain, for ERG’s leukemogenic activity. Methods: A genomic and proteomic analysis has been used to study the role of P199 in the leukemogenic pathway of ERG. Results: We specifically demonstrate in functional and gene expression assays that P199 is required for ERG-induced myeloid differentiation restriction and for self-renewal and maintenance of murine hematopoietic stem and progenitor cells (HSPC). As P199 is located within the PNT domain (protein interaction domain), we attempted to identify key protein interactions associated with leukemia progression induced by ERG. To this end, we used proximity ligation-mass spectrometry (BioID). In HEK293 cells, proximity maps of WT-ERG and a mutated form of ERG at position 199 (P199L-ERG) were compared. A total of 240 putative protein interactors with a significance analysis of interactome were identified. The hits were highly enriched with chromatin modifiers. Most significantly, the mutation severely impaired ERG’s interaction with components of the NCoR-HDAC3 complex, resulting in a 40% reduction in the number of spectral counts in comparison with wild-type ERG. ChIP sequencing for histone markers conducted on ER-Hoxb8 cells (transformed murine GMPs) demonstrated a decrease in H3K27ac signature at over 1500 unique sites in cells transduced with WT-ERG compared to those transduced with the mutant. Interestingly, these sites were predominantly associated with enhancers of genes expressed in mature myeloid cells. Next, we addressed the role of HDAC3 in ERG’s transcriptional activity. RNA sequencing of human ER-Hoxb8 cells treated with BRD3308 (an HDAC3 inhibitor) for 48 hours was performed. Interestingly, leukemia-associated pathways (Hoxa9-Meis1, CBFA2T3 and repression of myeloid differentiation genes) were ranked at the leading edge of gene sets that were significantly perturbed after HDAC3 inhibition. Moreover, we tested the significance of the ERG/NCoR-HDAC3 interaction in models of human leukemia. We demonstrated that ERG-dependent human AML cells as SKNO1 (AML1-ETO) and TF1 (CBFA2T3-ABDH12) were more sensitive to HDAC3 inhibition (RGFP966) as compared to ERG independent cells. Furthermore, RGFP966 exhibited a significant in-vivo antileukemic effect as measured by the burden of disease in the bone marrow of NSG mice transplanted with SKNO1 cells. By using the CRISPR-dCAS9 system we were able to show that leukemia development is abrogated in mice transplanted with SKNO1 cells expressing a reduced level of HDAC3. Remarkably, this effect was translated into a significant survival advantage. Summary/Conclusion: Taken together, our findings indicate that the aberrant overexpression of ERG maintains HSPCs in an undifferentiated state and promotes AML development. We suggest that the interaction between ERG and the NCoR-HDAC3 complex has an important role in the leukemogenic process, and that HDAC3 inhibition could be beneficial in AML characterized by high ERG expression.

In situ expression of ERG protein in the context of tumor heterogeneity identifies prostate cancer patients with inferior prognosis.

Prognostic biomarkers for prostate cancer are needed to improve prediction of disease course and guide treatment decisions. However, biomarker development is complicated by the common multifocality and heterogeneity of the disease. We aimed to determine the prognostic value of candidate biomarkers transcriptional regulator ERG and related ETS family genes, while considering tumor heterogeneity. In a multisampled, prospective, and treatment‐naïve radical prostatectomy cohort from one tertiary center (2010–2012, median follow‐up 8.1 years), we analyzed ERG protein (480 patients; 2047 tissue cores), and RNA of several ETS genes in a subcohort (165 patients; 778 fresh‐frozen tissue samples). Intra‐ and interfocal heterogeneity was identified in 29% and 33% (ERG protein) and 39% and 27% (ETS RNA) of patients, respectively. ERG protein and ETS RNA was identified exclusively in a nonindex tumor in 31% and 32% of patients, respectively. ERG protein demonstrated independent prognostic value in predicting biochemical (P = 0.04) and clinical recurrence (P = 0.004) and appeared to have greatest prognostic value for patients with Grade Groups 4–5. In conclusion, when heterogeneity is considered, ERG protein is a robust prognostic biomarker for prostate cancer.

Abstract 1851: Selective targeting of aberrant ETS transcription factors using uniquely modified mithramycin analogs

Abstract Background: Recent studies demonstrated that the aberrant oncogenic activity of ETS transcription factor fusions such as EWS-FLI1 and EWS-ERG in Ewing sarcoma can be targeted with the natural product mithramycin (MTM), which is known to have poor pharmacology. We hypothesized that the pharmacological properties of MTM can be ameliorated by rational semisynthetic modifications that improve its pharmacokinetic (PK) liabilities and expand its efficacy toxicity window. Further, we reasoned that the MTM core of each analog can facilitate DNA docking while the substitutions may interact uniquely with oncogenic ETS transcriptional complexes in different cancers. Here we present the pharmacologic properties of two analogs and their unique transcriptome profiles in TC32 Ewing sarcoma and VCaP prostate cancer cells, both of which depend on the aberrant oncogenic activity of an ETS gene fusion. Methods: MTM analogs were generated by semisynthetic conjugation to the 3-side chain of MTM. Cytotoxicity (GI50) was assessed using the resazurin assay following 72 hr incubation with test compounds (0.3nM-10uM). PK properties were assessed in athymic nu/nu mice after a bolus tail-vein injection and concentrations were assessed in plasma using LC/MS/MS methods. The maximum tolerated dose (MTD) was estimated based on weight loss and blood hematology. In vivo efficacy was assessed in subcutaneous xenografts established, which were dosed SIDx5 at the respective MTD or fractions of the MTD. Transcriptomic profiles were assessed in cells exposed to their respective 72 hr GI50 for 24 hr and in livers of mice that were dosed SIDx5. Results: Here we demonstrate that select modifications displayed unique cytotoxicity and efficacy Ewing sarcoma (EWS-FLI1) and VCaP prostate (TMPRSS2-ERG) cancer cell lines. Analogues with bulky amino acid substitutions in the 3-side chain of MTM had significantly improved PK in mice, as compared to MTM. Select analogs displayed selective efficacy in xenograft models of Ewing sarcoma at doses ranging as low as 1/3 of the MTD. Significantly, analogues of MTM displayed significantly different transcriptomic profiles in each cell line, which suggests that these molecules have unique properties despite sharing a core DNA-binding structure with MTM. Conclusions: MTM analogues with bulky hydrophobic amino acid substitutions on the 3-side chain showed the greatest improvement in PK properties and efficacy. Presumably PK are affected due to increased protein binding and an increase in the pKa, which leads to a lower ionized fraction and decreased uptake in the liver by organic anion transporters. This increase in plasma exposure may lead to increased drug partition in the tumor, as compared to MTM, and, therefore, improved efficacy. Transcriptomic data suggest that the analogs have unique transcriptional effects and therefore unlikely to manifest the same toxicity profile as MTM. Citation Format: Markos Leggas, Ambika Dudhate, Jurgen Rohr. Selective targeting of aberrant ETS transcription factors using uniquely modified mithramycin analogs [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 1851.

Abstract 462: Using attention-based deep multiple instance learning to identify key genetic alterations in prostate cancer from whole slide images

Abstract Prostate cancer (PCa) is associated with several genetic alterations which play an important role in the disease heterogeneity and clinical outcome. These alterations involve gene fusion between TMPRSS2 and members of the ETS family of transcription factors like ERG, ETV1, and ETV4 together with mutations or deletions in tumor suppressors like TP53 and PTEN. The expanding wealth of digital whole slide images (WSIs) and the increasing adoption of deep learning approaches offer a unique opportunity for pathologists to streamline the detection of these alterations. Here, we used 736 haematoxylin and eosin-stained WSIs from 494 primary PCa patients to identify several key genetic alterations including ERG, ETV1, and ETV4 fusion, PTEN loss, and TP53 and SPOP mutations. Using a custom segmentation pipeline, we identified tissue regions and tiled them into high-resolution (10X magnification) patches (256X256 pixels) which were passed to our deep multiple instance learning framework. Using a pre-trained ResNet50 model, we extracted informative features which were subsequently used for training to predict slide-level labels and to detect slide regions with high diagnostic relevance. Using a 10-folds cross validation approach, we divided the data into training (80%), validation (10%) and testing (10%) sets. The training and validation data were used for training the model and hyperparameters tuning, respectively while the testing data was used to provide an unbiased evaluation of the models’ performance using the mean Area Under the Receiver Operating Characteristic (AUROC) across the ten testing folds as evaluation metric. We managed to accurately detect key molecular alterations including ERG fusion, ETV1 fusion, ETV4 fusion, and PTEN loss. Additionally, we were able to detect mutations in TP53 and SPOP together with the presence of androgen-receptor splice variant 7 (ARv7). In addition to slide-level classification, we also identified subregions with high attention score which can help pathologists identify the distinct morphological features associated with each genetic alteration. Finally, in order to examine the cellular structure associated with each genetic alteration, we used Hover-Net model to segment and classify the nuclei in the high-attention tiles. Our work highlights the utility of using WSIs to accurately identify key molecular alteration in cancer and their associated morphological and cellular features on the slide which would streamline the diagnostic process. To the best of our knowledge, this is the first study that uses routine WSIs to predict and characterize key genetic alterations in PCa. Citation Format: Mohamed Omar, Zhuoran Xu, Ryan Carelli, Jacob Rosenthal, David Brundage, Daniela C. Salles, Eddie L. Imada, Renato Umeton, Edward M. Schaeffer, Brian D. Robinson, Tamara L. Lotan, Massimo Loda, Luigi Marchionni. Using attention-based deep multiple instance learning to identify key genetic alterations in prostate cancer from whole slide images [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 462.

Abstract 1471: Over-expression of WW domain containing transcription regulator 1 (TAZ/WWTR1) in prostate cancer and its modulation by prostate-derived ETS factor (PDEF)

Abstract Objective: WW domain containing transcription regulator 1 (TAZ/WWTR1), one of the two downstream effectors of the Hippo pathway, is known to regulate cancer cell proliferation, migration and apoptosis by acting as a transcriptional co-activator. Recently, we described altered expression of yes-associated protein (YAP1) (a paralog of TAZ) in prostate cancer. However, the expression and function of TAZ and its regulation in prostate cancer is not well characterized. In the present study, we profiled expression patterns of TAZ in clinical data sets of PCa and evaluated the effects of PDEF on TAZ phosphorylation in prostate cancer (PCa) cells in culture. Material & Method: We analyzed the publicly available PCa datasets through Ualcan and c-Bioportal webservers. We transfected prostate cancer PC3 cells with PDEF or respective vector control. Protein levels of TAZ were analyzed by western blotting. Global changes in gene expression were analyzed using Affymetrix arrays followed by Gene Set Enrichment analysis. Results: Our analysis of publicly available PCa patient data in TCGA dataset, 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 during disease progression and metastasis. Our analysis of data sets in c-Bio portal revealed that patients with neuroendocrine PCa harbor much higher levels of TAZ mRNA as compared to CRPC phenotype. We also observed a negative correlation between PDEF and TAZ expression in CRPC/NEPC data set. We observed that the PC3 cells transfected with PDEF (PDEF-PC3) showed an increased phospho-TAZ as compared to the respective PC3 vector control (VC-PC3) suggesting that PDEF plays a critical role in modulating TAZ activity an important effector in Hippo signaling. Conclusions: These results provide first direct demonstration of elevated expression of TAZ in PCa, suggesting potential role of TAZ in prostate cancer. We also show for the first time increased TAZ phosphorylation in PCa cells by PDEF. Taken together with our recent work, our data suggest that PDEF modulates Hippo signaling by targeting both YAP1 and TAZ in PCa. Citation Format: Praveen K. Jaiswal, Suman Mohajan, Sweaty Koul, Hari K. Koul. Over-expression of WW domain containing transcription regulator 1 (TAZ/WWTR1) in prostate cancer and its modulation by prostate-derived ETS factor (PDEF) [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 1471.

Abstract LB036: Molecular insight into ERG transcriptional activity to unravel novel therapeutic options for ERG-positive castration resistant prostate cancer

Abstract Gene fusions of the AR-regulated gene TMPRSS2 with the ETS family transcription factor ERG characterize about 40% of castration resistant prostate cancer (CRPC) - an advanced and lethal stage of prostate cancer. AR-mediated overexpression of ERG hijacks AR inhibiting lineage-specific differentiation and increases cell invasion and migration. In vivo work revealed a causal role of ERG in initiating prostate cancer. Notably, in vitro and in vivo models of CRPC endogenously bearing T2ERG depend on ERG activity for proliferation and survival. Thus, pursuing the development of ERG-based therapeutic strategies represents a promising route for CRPC treatment, although major challenges in targeting transcription factors such as ERG have been reported. Yet, synthetic lethality approaches including genome-wide loss-of-function screens can be employed to avoid direct ERG targeting. Here, we used metastatic PCa cell lines (LNCaP, 22Rv1 and DU145) engineered with inducible systems to overexpress ERG to show that high levels of ERG expression lead to an apparent reduction in the fitness of AR-positive and negative cells. Prolonged overexpression of a mutant form of ERG, which is not able to bind DNA, does not affect cell viability indicating that ERG DNA binding and transcriptional activity are necessary for the induction of this phenotype. The ERG-mediated reduction in cellular fitness is influenced by cell adhesion and is characterized by cell cycle arrest mediated by several cell cycle inhibitors induced by ERG. As revealed by gene expression analyses and functional assays, ERG induces a less proliferative, more dedifferentiated and invasive phenotype, which lead to an apparent reduction of cellular fitness in vitro, but is characteristic of more aggressive tumor cells in vivo. Altogether, these results underscore the reliability of the developed in vitro models for ERG activity, which will be used for screening to identify therapeutic vulnerabilities unlocked by ERG in CRPC. Importantly, we also uncovered confounding factors (i.e. loss of ERG-positive cells not due to screen selection) that could impinge on the results arising from in vitro screens. Citation Format: Francesca Lorenzin, Giulia Fracassi, Yari Ciani, Paola Gasperini, David Rickman, Francesca Demichelis. Molecular insight into ERG transcriptional activity to unravel novel therapeutic options for ERG-positive castration resistant prostate cancer [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 LB036.

Abstract LB190: Modulation of SPI1 transcriptional program contributes to the preclinical anti-tumor activity of SMARCA4/SMARCA2 ATPase inhibitors in AML

Abstract SPI1 (PU.1) is an ETS family transcription factor that plays a critical role in hematopoietic development and differentiation. Regulation of SPI1 expression has also been implicated in Acute Myeloid Leukemia (AML) oncogenesis, though its mechanism is incompletely understood. Previously we have identified and characterized a series of novel dual inhibitors of the SMARCA4/SMARCA2 ATPases (also referred to as BRG1/BRM), critical components of the BAF (mSWI/SNF) family of chromatin remodeling complexes; and FHD-286, a related SMARCA4/SMARCA2 ATPase inhibitor, is currently being explored clinically for the treatment of metastatic uveal melanoma and AML (NCT04879017 and NCT04891757). Here we show that AML cell lines are sensitive to BAF ATPase inhibition, resulting in both cell cycle arrest and apoptosis. We demonstrate that BAF ATPase inhibition primarily affects the SPI1 transcriptional profile by regulating SPI1 genomic occupancy at various enhancer elements, resulting in downregulation of key target genes. Using in vivo mouse models of AML, we demonstrate dose-dependent tumor growth inhibition and pharmacodynamic modulation of SPI1 target genes. Together, these data suggest that modulation of SPI1 function may, in part, provide a mechanistic basis for the clinical development of FHD-286 in AML. Citation Format: Gabriel J. Sandoval, Katharine Feldman, Sal Topal, Ammar Adam, Hsin-Jung Wu, Darshan Sappal, Luis M. Soares, David L. Lahr, Lan Xu, Rishi G. Vaswani, Jordana Muwanguzi, Liyue Huang, Jessica Piel, Mike Collins, Ho Man Chan, Michael J. Thomenius, Steven F. Bellon, Ryan G. Kruger, Carl P. Decicco, Richard C. Centore, Martin Hentemann. Modulation of SPI1 transcriptional program contributes to the preclinical anti-tumor activity of SMARCA4/SMARCA2 ATPase inhibitors in AML [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 LB190.

Abstract 2365: Mechanisms of mutant p53 targeting to the genome

Abstract Mutations in genes often result in tumor formation, especially when these mutations occur in tumor suppressors. TP53 is a tumor suppressor that is frequently mutated in cancer, with mutations in this gene occurring in nearly 50% of all cancer cases. Once mutated, p53 loses its tumor suppressive function while simultaneously gaining oncogenic function. One of the functions that mutant p53 loses is the ability to directly bind to chromatin, however, it has been reported that mutant p53 can still affect the transcriptome of cancer cells via interactions with other transcription factors. One of these interacting partners is ETS2. ETS2 belongs to the ETS transcription factor family, which has 28 family members. This family is characterized by their affinity for an ETS binding site (EBS). EBS’s are present in 50% of all mutant p53 occupied promoters. Other ETS family members have also been linked to mutant p53 but these interactions have either been deemed as weak (ETS1) or have yet to be identified as direct (ERG). To determine which ETS proteins interact with mutant p53 I conducted affinity pull-down assays using purified ETS proteins and purified mutants of p53. My data shows that several ETS proteins interact with mutant p53 better than ETS2. I then sought to determine which residues are important for this interaction through truncation studies in which I used purified truncations of ETS proteins and purified mutants of p53. I found that ERG, one protein that strongly interacted with mutant p53, had two interaction interfaces. This may explain why the interaction is strong. My next step was to determine which ETS proteins are responsible for the targeting of mutant p53 to the genome. To address the requirement for ETS to recruit mutant p53 to chromatin I performed chromatin immunoprecipitation sequencing studies in the presence or absence of different ETS factors to determine differences in mutant p53 binding. For these studies I knocked down ETS2 or ERG prior to performing p53 ChIP-Seq to determine differences in p53 binding to chromatin under these conditions. My analyses of these data indicate that each of the conditions resulted in different p53 binding patterns in the ChIP-Seq and that there is a requirement for ETS in mutant p53 binding.My studies have demonstrated that ETS proteins interact with mutant p53 and that this interaction seems to be required for mutant p53 binding to the genome. My future work will test phenotypes related to ETS/mutant p53 interactions. Ultimately, if ETS/mutant p53 interactions are deemed important for oncogenic function, these will be attractive targets for future drug development. Citation Format: Stephanie Metcalf, Stevie Morris, Peter Hollenhorst. Mechanisms of mutant p53 targeting to the genome [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 2365.

Abstract 2363: Transcription factors of keratin 18 gene in MDA-MB-231Br (brain-colonizing) breast cancer cells

Abstract Among all cancer types, breast cancer has the highest incidence rate and second highest mortality rate in females in the United States. Breast cancer patients with brain metastasis have a poor prognosis, with a median survival time of less than 1 year despite treatment. Decreasing the prevalence of brain metastasis would improve the overall prognosis of patients. We previously built a novel mouse model of breast cancer brain metastasis by intracardiac injection of the MDA-MB-231 (triple-negative breast cancer cell line) cells in mice. The brain colonizing counterpart of the parental cells primarily metastasizes to the brain, which is called the MDA-MB-231Br cell line. We have also shown that keratin 18 (an epithelial marker) plays a crucial role in the brain metastasis process as the MDA-MB-231Br cells have lower expression of the keratin 18 gene compared to their parental MDA-MBA-231 cells. Therefore, it is important to understand the transcriptional control of the keratin 18 gene, in order to fully understand the role of keratin 18 in regulating brain metastasis of breast cancer. We hypothesize that keratin 18 gene expression differs between these two cell lines due to the differential occupation of the gene by transcription factors such as Fli-1 or ETS-1. In this study, we analyzed the pattern of chromatin occupation of transcription factors ETS-1 and Fli-1 to determine whether they are responsible for the altered expression of the keratin 18 gene in MDA-MB-231Br cells.The Chromatin Immunoprecipitation (ChIP) was performed using the Pierce Magnetic ChIP Kit to analyze the presence of transcription factors Fli-1 and ETS-1 with enzymatic and mechanical shearing combined. The ChIP Kit included the positive control anti-RNA Polymerase II Antibody and GAPDH control primers. We optimized the experimental conditions including ChIP analysis for MDA-MB-231Br cells, detection of expected positive control results, and sonication settings to break apart the chromatin. We measured the ChIP mediated enrichment of the intron 1 region of the keratin 18 gene, as the DNA is hypermethylated in this region in MDA-MB-231Br cells. The primers were designed and used as follows: forward: 5′-GATCATCGAGGACCTGAGGG-3′ and reverse: 5′-GGGGAGCAGATCCTTCTTAGC-3′. Our results indicated that an ETS transcription factor occupies intron 1 of the keratin 18 gene in the MDA-MB-231Br cells, also showing that ChIP is an effective method to examine transcription factor binding. For future studies, we plan to perform similar experiments with the parental MDA-MB-231 cells. Then a quantitative PCR will be used to measure DNA binding sites of the transcription factors and determine if there is a significant difference in the level of chromatin occupation between the regular and brain-colonizing breast cancer cells. Our studies are necessary to understand the role of the keratin 18 gene in regulating tumor metastasis at the molecular level. Citation Format: Anissa Elice Johnson, Sagardeep Singh, Paul Lockman, Tuoen Liu, Gabor Szalai. Transcription factors of keratin 18 gene in MDA-MB-231Br (brain-colonizing) breast cancer cells [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 2363.