Ets Proteins Research Articles

The Role of Pea3 Transcription Factor Subfamily in the Nervous System.

ETS domain transcription factor superfamily is highly conserved throughout metazoa and is involved in many aspects of development and tissue morphogenesis, and as such, the deregulation of ETS proteins is quite common in many diseases, including cancer. The PEA3 subfamily in particular has been extensively studied with respect to tumorigenesis and metastasis; however, they are also involved in the development of many tissues with branching morphogenesis, such as lung or kidney development. In this review, we aim to summarize findings from various studies on the role of Pea3 subfamily members in nervous system development in the embryo, as well as their functions in the adult neurons. We further discuss the different signals that were shown to regulate the function of the Pea3 family and indicate how this signal-dependent regulation of Pea3 proteins can generate neuronal circuit specificity through unique gene regulation. Finally, we discuss how these developmental roles of Pea3 proteins relate to their role in tumorigenesis.

Potential influence of MIR-204-3P on ETS-1 protein expression in papillary thyroid carcinoma

Potential influence of MIR-204-3P on ETS-1 protein expression in papillary thyroid carcinoma

Mesenchymal Stem Cell-Derived Exosomes Mitigate Acute Murine Liver Injury via Ets-1 and Heme Oxygenase-1 Up-regulation.

Mesenchymal stem cells (MSCs)-derived exosomes have been previously demonstrated to promote tissue regeneration in various animal disease models. This study investigated the protective effect of exosome treatment in carbon tetrachloride (CCl4)-induced acute liver injury and delineated possible underlying mechanism. Exosomes collected from conditioned media of previously characterized human umbilical cord-derived MSCs were intravenously administered into male CD-1 mice with CCl4-induced acute liver injury. Biochemical, histological and molecular parameters were used to evaluate the severity of liver injury. A rat hepatocyte cell line, Clone-9, was used to validate the molecular changes by exosome treatment. Exosome treatment significantly suppressed plasma levels of AST, ALT, and pro-inflammatory cytokines, including IL-6 and TNF-α, in the mice with CCl4-induced acute liver injury. Histological morphometry revealed a significant reduction in the necropoptic area in the injured livers following exosome therapy. Consistently, western blot analysis indicated marked elevations in hepatic expression of PCNA, c-Met, Ets-1, and HO-1 proteins after exosome treatment. Besides, the phosphorylation level of signaling mediator JNK was significantly increased, and that of p38 was restored by exosome therapy. Immunohistochemistry double staining confirmed nuclear Ets-1 expression and cytoplasmic localization of c-Met and HO-1 proteins. In vitro studies demonstrated that exosome treatment increased the proliferation of Clone-9 hepatocytes and protected them from CCl4-induced cytotoxicity. Kinase inhibition experiment indicated that the exosome-driven hepatoprotection might be mediated through the JNK pathway. Exosome therapy activates the JNK signaling activation pathway as well as up-regulates Ets-1 and HO-1 expression, thereby protecting hepatocytes against hepatotoxin-induced cell death.

Abstract PO-015: The RNA helicase DDX21 cooperates with ETS1 and FLI1 in cell cycle regulation, immune evasion and small nucleolar RNA processing to sustain the survival of DLBCL cells

Abstract Introduction.The ETS1 and FLI1 transcription factors are both located in the 11q24.3 region, which is commonly amplified in 25% of diffuse large B cell lymphomas (DLBCL) (Bonetti et al, 2013) and largely co-regulate a series of genes involved in B-cell signaling, differentiation and cell cycle (Priebe et al, 2020; Sartori et al, 2021). While FLI1 is expressed at a higher level in DLBCL of the germinal center B-cell (GCB) type than in the activated B-cell-like (ABC) DLBCL, ETS1 is more expressed in the latter subgroup. We and others have reported preclinical anti-tumor activity in lymphomas and other tumors with small molecules blocking the binding of ETS factors and RNA helicases (Erkizan et al, 2009; Spriano et al, 2019). Methods.To explore interaction partners, protein precipitation was conducted utilizing streptavidin beads to capture strep-tagged ETS1 in the ABC DLBCL cell line, HBL-1, followed by Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS). Shotgun proteomics analysis identified biologically relevant ETS1 protein interactors, with a spectral count above four, that were validated by normal and reverse co-immunoprecipitation experiments. Transcriptome analysis was done via RNASeq and smallRNASeq after DDX21 siRNAs silencing in four DLBCL cell lines, two derived from ABC (HBL1 and U2932) and two from GCB (OCI-Ly1 and VAL). Direct targets were validated by DDX21 chromatin immunoprecipitation (ChIP). Results. The ETS1 interactors included proteins associated with RNA processing. Among these, we focused on the novel interactor DDX21, an RNA helicase also regulated by pETS1 (Chung et al, 2023) and FLI1 (Sartori et al, 2021). DDX21 was found differentially expressed between GCB and ABC DLBCL, with higher expression in the latter (P<0.001). When we silenced DDX21 with siRNAs, toxicity was seen in ABC cell lines. To better characterize the role of DDX21 as a transcriptional factor in DLBCL we obtained RNASeq transcriptome after DDX21 silencing. Transcriptional profiling and functional annotation of transcripts regulated by DDX21 revealed genes coding proteins involved in cell cycle (FDR <0.001), ribosomes (FDR <0.001) and immune evasion (FDR <0.001). Based on the role of DDX21, smallRNAseq was also performed and miRNAs of some members of the let7 family (hsa-let-7d, hsa-let-7e and hsa-let-7f) were found downregulated by DDX21, together with miR-532. Among the upregulated miRNAs miR-222 and miR-155 were the most interesting. Different snoRNAs were also found deregulated, SNORA37 was further validated as a DDX21 upregulated direct target by RNA and ChIP qPCR. Conclusions. In ABC DLBCL, ETS1 interacts with proteins involved in spliceosome and in ribosome biogenesis, including DDX21. More expressed in ABC than GCB DLBCL, DDX21 appears essential for the survival of ABC lymphoma cells by regulating cell cycle, RNA processing and immune evasion. Several miRNAs and snoRNAs were found deregulated by DDX21. The interaction between ETS1 and DDX21 offers an additional therapeutic opportunity against DLBCL cells. Citation Format: Giulio Sartori, Valdemar Priebe, Luciano Cascione, Elaine Y.L. Chung, Mélanie Favre-Juilland, Sara Napoli, Alberto Arribas, Stefano Giuffrida, Andrea Rinaldi, Margot Thome Miazza, Francesco Bertoni. The RNA helicase DDX21 cooperates with ETS1 and FLI1 in cell cycle regulation, immune evasion and small nucleolar RNA processing to sustain the survival of DLBCL cells [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-015.

Mechanoresponsive ETS1 causes endothelial dysfunction and arterialization in varicose veins via NOTCH4/DLL4 signaling

Varicose veins are the most common venous disorder in humans and are characterized by hemodynamic instability due to valvular insufficiency and orthostatic lifestyle factors. It is unclear how changes in biomechanical signals cause aberrant remodeling of the vein wall. Our previous studies suggest that Notch signaling is implicated in varicose vein arterialization. In the arterial system, mechanoresponsive ETS1 is a transcriptional activator of the endothelial Notch, but its involvement in sensing disrupted venous flow and varicose vein formation has not been investigated. Here, we use human varicose veins and cultured human venous endothelial cells to show that disturbed venous shear stress activates ETS1-NOTCH4/DLL4 signaling. Notch components were highly expressed in the neointima, whereas ETS1 was upregulated in all histological layers of varicose veins. In vitro microfluidic flow-based studies demonstrate that even minute changes in venous flow patterns enhance ETS1-NOTCH4/DLL4 signaling. Uniform venous shear stress, albeit an inherently low-flow system, does not induce ETS1 and Notch proteins. ETS1 activation under altered flow was mediated primarily by MEK1/2 and, to a lesser extent, by MEK5 but was independent of p38 MAP kinase. Endothelial cell-specific ETS1 knockdown prevented disturbed flow-induced NOTCH4/DLL4 expression. TK216, an inhibitor of ETS-family, prevented the acquisition of arterial molecular identity and loss of endothelial integrity in cells exposed to the ensuing altered shear stress. We conclude that ETS1 senses blood flow disturbances and may promote venous remodeling by inducing endothelial dysfunction. Targeting ETS1 rather than downstream Notch proteins could be an effective and safe strategy to develop varicose vein therapies.

Oncogenic ETS fusions promote DNA damage and proinflammatory responses via pericentromeric RNAs in extracellular vesicles.

Aberrant expression of the E26 transformation-specific (ETS) transcription factors characterizes numerous human malignancies. Many of these proteins, including EWS:FLI1 and EWS:ERG fusions in Ewing sarcoma (EwS) and TMPRSS2:ERG in prostate cancer (PCa), drive oncogenic programs via binding to GGAA repeats. We report here that both EWS:FLI1 and ERG bind and transcriptionally activate GGAA-rich pericentromeric heterochromatin. The respective pathogen-like HSAT2 and HSAT3 RNAs, together with LINE, SINE, ERV, and other repeat transcripts, are expressed in EwS and PCa tumors, secreted in extracellular vesicles (EVs), and are highly elevated in plasma of patients with EwS with metastatic disease. High human satellite 2 and 3 (HSAT2,3) levels in EWS:FLI1- or ERG-expressing cells and tumors were associated with induction of G2/M checkpoint, mitotic spindle, and DNA damage programs. These programs were also activated in EwS EV-treated fibroblasts, coincident with accumulation of HSAT2,3 RNAs, proinflammatory responses, mitotic defects, and senescence. Mechanistically, HSAT2,3-enriched cancer EVs induced cGAS-TBK1 innate immune signaling and formation of cytosolic granules positive for double-strand RNAs, RNA-DNA, and cGAS. Hence, aberrantly expressed ETS proteins derepress pericentromeric heterochromatin, yielding pathogenic RNAs that transmit genotoxic stress and inflammation to local and distant sites. Monitoring HSAT2,3 plasma levels and preventing their dissemination may thus improve therapeutic strategies and blood-based diagnostics.

Abstract 1706: Investigating the interaction between ETS family members and mutant p53

Abstract Cancer cases are on the rise globally requiring a deeper understanding of the disease and identification of novel therapeutic targets. Mutations in genes often result in tumor formation. TP53 encodes p53, a tumor suppressive transcription factor, and is mutated in nearly 50% of all cancer cases. Once mutated, p53 loses its tumor suppressive function while simultaneously gaining oncogenic function. p53 mutations disrupt direct p53 binding to DNA and promotes p53 binding to regulatory regions of oncogenes via protein-protein interactions with other transcription factors. One of these interacting partners is ETS2, a member of the ETS transcription factor family. ETS binding sites are present in 50% of all mutant p53 occupied regulatory elements. Other ETS family members have also been linked to mutant p53 but these interactions have either been deemed weak (ETS1) or have yet to be identified as direct (ERG). It is my aim to determine the scope of interaction between the ETS family and mutant p53 and to elucidate whether this interaction is required for the observed oncogenic phenotypes of ETS proteins and mutant p53.To determine which ETS proteins interact with mutant p53 purified ETS proteins were used for affinity pull-down of purified mutant p53. The entire ETS family interacted with mutant p53 to some degree, and several ETS proteins had stronger interactions than ETS2. Truncation studies were used to determine interaction domains. One interacting region was the conserved DNA binding domain, which may explain interaction across the entire family. ERG and ETS2, both strong mutant p53 interactors, had two interaction interfaces. I hypothesize that the second interaction interface defines strong interactors. To determine if interacting ETS are responsible for targeting mutant p53 to the genome, chromatin immunoprecipitation sequencing measured mutant p53 binding in the presence or absence of different ETS factors. These data indicate that each of the conditions resulted in different p53 binding patterns and that there is a requirement for ETS in mutant p53 binding. Lastly, I wanted to determine if there was a correlation between p53 mutation and expression of strong mutant p53 interactors in certain cancer types. Analysis of TCGA samples showed that in ovarian cancers with p53 mutations, ETS factors that are strong mutant p53 interactors were upregulated in a mutually exclusive pattern, while weak interactors tended to be downregulated, or unchanged. Similar trends were observed among some other cancer types. My studies have shown that several ETS proteins interact with mutant p53 in vitro, recruit p53 to the genome, and that this interaction pattern correlates with expression in mutant p53 driven cancers. Additionally, studies are ongoing to determine phenotypes related to this interaction. Ultimately, if ETS/mutant p53 interactions are deemed important for oncogenesis, these will be attractive targets for drug development. Citation Format: Stephanie Metcalf, Peter Hollenhorst. Investigating the interaction between ETS family members and mutant p53 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1706.

Abstract 3033: Oncogenic ETS transcription factors avoid repression by EZH2 and FOXO1 in prostate cancer and Ewing sarcoma

Abstract ETS transcription factors play roles throughout development, aiding in hematopoiesis, blood vessel formation and cell fate. The ETS family is composed of 28 members, all of which share an ETS DNA-binding domain. Chromosomal rearrangements that lead to the overexpression of specific ETS promote oncogenic transformation and tumor development. More than half of prostate tumors are driven by aberrant expression of an ETS protein. Our lab has demonstrated that oncogenic ETS form essential interactions with a ubiquitous RNA-binding protein, EWS, to promote prostate tumorigenesis. A similar mechanism exists in Ewing sarcoma (ES). ES tumors are driven by the expression EWS/ETS fusion proteins. This project focuses on the similarities and differences between oncogenic ETS in prostate cancer and EWS/ETS fusions in Ewing sarcoma. We compared biological functions of the most common oncogenic ETS in prostate cancer, ERG, and the most common fusion in ES, EWS/FLI1. Both ERG and EWS/FLI1 promoted migration and clonogenic survival in normal prostate epithelial cell lines, RWPE1 and PNT2. These data suggest that the EWS-ERG complex and EWS/FLI1 fusion protein can function similarly in prostate cells. Knockdown of endogenous EWS/FLI1 in the ES cell line, A673, reduced anchorage-independent colony formation in soft agar. Rescue with exogenous EWS/FLI1 or EWS/ERG restored colony formation relative to control. Wildtype ERG was unable to rescue colony formation in A673. This suggests that ERG and EWS/FLI1 function differently in A673 cells. Pulldown assays from A673 lysates reveal that ERG interacts with EZH2 and FOXO1. Our lab has established that ERG acts as a transcriptional repressor through the formation of an ERG-EZH2-PRC2 complex. Others demonstrate that FOXO1 represses ERG activity by reducing its recruitment to the target sites. Both the N-terminal truncation of ERG and phosphorylation of ERG at S96 disrupted these interactions and rescued anchorage independent growth in A673 ES cells. These data suggest that EWS/ETS fusions avoid transcriptional repression in Ewing tumors due to loss of the N-terminus of the ETS protein. Citation Format: Nicholas F. Downing, Kaitlyn M. Mills, Peter C. Hollenhorst. Oncogenic ETS transcription factors avoid repression by EZH2 and FOXO1 in prostate cancer and Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3033.

Genome-wide identification and characteristic analysis of ETS gene family in blood clam Tegillarca granosa

BackgroundETS transcription factors, known as the E26 transformation-specific factors, assume a critical role in the regulation of various vital biological processes in animals, including cell differentiation, the cell cycle, and cell apoptosis. However, their characterization in mollusks is currently lacking.ResultsThe current study focused on a comprehensive analysis of the ETS genes in blood clam Tegillarca granosa and other mollusk genomes. Our phylogenetic analysis revealed the absence of the SPI and ETV subfamilies in mollusks compared to humans. Additionally, several ETS genes in mollusks were found to lack the PNT domain, potentially resulting in a diminished ability of ETS proteins to bind target genes. Interestingly, the bivalve ETS1 genes exhibited significantly high expression levels during the multicellular proliferation stage and in gill tissues. Furthermore, qRT-PCR results showed that Tg-ETS-14 (ETS1) is upregulated in the high total hemocyte counts (THC) population of T. granosa, suggesting it plays a significant role in stimulating hemocyte proliferation.ConclusionOur study significantly contributes to the comprehension of the evolutionary aspects concerning the ETS gene family, while also providing valuable insights into its role in fostering hemocyte proliferation across mollusks.

DNA selection by the master transcription factor PU.1

The master transcriptional regulator PU.1/Spi-1 engages DNA sites with affinities spanning multiple orders of magnitude. To elucidate this remarkable plasticity, we have characterized 22 high-resolution co-crystallographic PU.1/DNA complexes across the addressable affinity range in myeloid gene transactivation. Over a purine-rich core (such as 5'-GGAA-3') flanked by variable sequences, affinity is negotiated by direct readout on the 5' flank via a critical glutamine (Q226) sidechain and by indirect readout on the 3' flank by sequence-dependent helical flexibility. Direct readout by Q226 dynamically specifies PU.1's characteristic preference for purines and explains the pathogenic mutation Q226E in Waldenström macroglobulinemia. The structures also reveal how disruption of Q226 mediates strand-specific inhibition by DNA methylation and the recognition of non-canonical sites, including the authentic binding sequence at the CD11b promoter. A re-synthesis of phylogenetic and structural data on the ETS family, considering the centrality of Q226 in PU.1, unifies the model of DNA selection by ETS proteins.

Sculponeatin A promotes the ETS1-SYVN1 interaction to induce SLC7A11/xCT-dependent ferroptosis in breast cancer

BackgroundE26 transformation specificity-1 (ETS1) is a transcription factor that is overexpressed in breast cancer (BC) and promotes tumor progression. Sculponeatin A (stA), a new diterpenoid extracted from Isodon sculponeatus, has no reported antitumor mechanism. PurposeHere, we explored the antitumor activity of stA in BC and further clarified its mechanism. MethodsFerroptosis was detected by flow cytometric, glutathione, malondialdehyde, and iron determination assays. The effect of stA on the upstream signaling pathway of ferroptosis was detected by Western blot, gene expression, gene alterations and other approaches. The binding of stA and ETS1 was examined through a microscale thermophoresis assay and a drug affinity responsive target stability assay. An in vivo mouse model experiment was performed to evaluate the therapeutic and potential mechanism of stA. ResultsstA exhibits therapeutic potential in BC by inducing SLC7A11/xCT-dependent ferroptosis. stA decreases the expression of ETS1, which is responsible for xCT-dependent ferroptosis in BC. stA inhibits the transcriptional expression of xCT by directly binding to the ETS domain of the ETS1 protein. In addition, stA promotes proteasomal degradation of ETS1 by triggering ubiquitin ligase synoviolin 1 (SYVN1)-mediated ubiquitination. The K318 site of ETS1 mediates ubiquitination of ETS1 by SYVN1. In a mouse model, stA inhibits tumor growth without causing obvious toxicity. ConclusionTaken together, the results confirm that stA promotes the ETS1-SYVN1 interaction to induce ferroptosis in BC mediated by ETS1 degradation. stA is expected to be used in research of candidate drugs for BC and drug design based on ETS1 degradation.

Nervous system-related gene regulatory networks and functional evolution of ETS proteins across species

The ETS domain transcription factor family is one of the major transcription factor superfamilies that play regulatory roles in development, cell growth, and cancer progression. Although different functions of ETS member proteins in the nervous system have been demonstrated in various studies, their role in neuronal cell differentiation and the evolutionary conservation of its target genes have not yet been extensively studied. In this study, we focused on the regulatory role of ETS transcription factors in neuronal differentiation and their functional evolution by comparative transcriptomics. In order to investigate the regulatory role of ETS transcription factors in neuronal differentiation across species, transcriptional profiles of ETS members and their target genes were investigated by comparing differentially expressed genes and gene regulatory networks, which were analyzed using human, gorilla, mouse, fruit fly and worm transcriptomics datasets. Bioinformatics approaches to examine the evolutionary conservation of ETS transcription factors during neuronal differentiation have shown that ETS member proteins regulate genes associated with neuronal differentiation, nervous system development, axon, and synaptic regulation in different organisms. This study is a comparative transcriptomic study of ETS transcription factors in terms of neuronal differentiation using a gene regulatory network inference algorithm. Overall, a comparison of gene regulation networks revealed that ETS members are indeed evolutionarily conserved in the regulation of neuronal differentiation. Nonetheless, ETS, PEA3, and ELF subfamilies were found to be relatively more active transcription factors in the transcriptional regulation of neuronal differentiation.

Identification and characterization of novel ETV4 splice variants in prostate cancer

ETV4, one of ETS proteins overexpressed in prostate cancer, promotes migration, invasion, and proliferation in prostate cells. This study identifies a series of previously unknown ETV4 alternatively spliced transcripts in human prostate cell lines. Their expression has been validated using several unbiased techniques, including Nanopore sequencing. Most of these transcripts originate from an in-frame exon skipping and, thus, are expected to be translated into ETV4 protein isoforms. Functional analysis of the most abundant among these isoforms shows that they still bear an activity, namely a reduced ability to promote proliferation and a residual ability to regulate the transcription of ETV4 target genes. Alternatively spliced genes are common in cancer cells: an analysis of the TCGA dataset confirms the abundance of these novel ETV4 transcripts in prostate tumors, in contrast to peritumoral tissues. Since none of their translated isoforms have acquired a higher oncogenic potential, such abundance is likely to reflect the tumor deranged splicing machinery. However, it is also possible that their interaction with the canonical variants may contribute to the biology and the clinics of prostate cancer. Further investigations are needed to elucidate the biological role of these ETV4 transcripts and of their putative isoforms.

ERG Functionally Overlaps with Other Ets Proteins in Promoting TH9 Cell Expression of Il9 during Allergic Lung Inflammation.

CD4+ TH cells develop into subsets that are specialized in the secretion of particular cytokines to mediate restricted types of inflammation and immune responses. Among the subsets that promote development of allergic inflammatory responses, IL-9-producing TH9 cells are regulated by a number of transcription factors. We have previously shown that the E26 transformation-specific (Ets) family members PU.1 and Ets translocation variant 5 (ETV5) function in parallel to regulate IL-9. In this study we identified a third member of the Ets family of transcription factors, Ets-related gene (ERG), that mediates IL-9 production in TH9 cells in the absence of PU.1 and ETV5. Chromatin immunoprecipitation assays revealed that ERG interaction at the Il9 promoter region is restricted to the TH9 lineage and is sustained during murine TH9 polarization. Knockdown or knockout of ERG during murine or human TH9 polarization in vitro led to a decrease in IL-9 production in TH9 cells. Deletion of ERG in vivo had modest effects on IL-9 production in vitro or in vivo. However, in the absence of PU.1 and ETV5, ERG was required for residual IL-9 production in vitro and for IL-9 production by lung-derived CD4 T cells in a mouse model of chronic allergic airway disease. Thus, ERG contributes to IL-9 regulation in TH9 cells.

The Transcription Factor ETS-1 Correlates with the Increased Age Associated B Cells in Patients with Acquired Aplastic Anemia

The increased IFN-g levels observed in acquired aplastic anemia (AA), have a crucial role in the immune destruction of the hematopoietic progenitors which subsequently lead to the clinical presentation of this rare and complicated disease. We and others have previously shown that the transcription factor T-bet is over-expressed in T cells from patients with AA. Recently it was shown that a subpopulation of B cells also express T-bet; these cells are characterized as age-associated B cells (ABCs) and express high levels of CD11c and CD19, and are negative for CD21. Stimulation of B cells leads to the formation of ABCs, which in turn "talk" to the T cells and stimulate them, leading to IFN-g production. IFN-g may lead to further induction of T-bet expressing B cells (ABCs). Ets-1 is an essential cofactor for STAT1 in the regulation of T-bet in ABCs. A small subset of CD4+helper T cells, circulating T follicular helper cells (Tfh) are also implicated in T-bet expression in ABCs. They are characterized by the surface expression of CXCR5, ICOS, and PD1, the transcription factor Bcl-6, and produce mainly IL-21, but also IL-17, IL-4, and IFN-γ. To date, there is no information on the expression of ABCs and Tfh cells in patients with aplastic anemia. The current study population included 19 patients with AA and 20 healthy volunteers. The diagnosis of AA was based on the criteria of the International Agranulocytosis and Aplastic Anemia Study. Marrow cytogenetics were normal in all aplastic anemia cases and Fanconi anemia was excluded in all patients. Peripheral blood samples were obtained after written informed consent and using protocols approved by the institutional review board of the University of Patras Medical School, Greece. At the time of sampling, 17 out of 19 of the patients included in the study were at initial diagnosis before receiving any treatment and two patients were in complete remission and off any immunosuppressive treatment. The ABCs, Tfh, and regulatory T cell populations were examined using flow cytometry. We first measured the frequency of ABCs (results are in % ± SEM). All patients had significantly increased numbers of ABCs compared to healthy volunteers (3.68 ± 0.82% vs 0.94 ± 0.34%, p=0.022, Figure 1A). In the two cases in remission, the ABCs levels were lower than the levels of patients at diagnosis. We further examined if there were any differences in the percentages of T follicular helper cells; Patients also showed increased levels of Tfh cells compared to healthy controls (4.02± 1.01% vs 1.11 ± 0.10%, p=0.023, Figure 1A). The patients showing increased levels of ABCs and Tfh cells were also examined for the presence of regulatory T cells (Tregs) as determined by the expression of CD4+ CD25hi , CD127low expression. As expected patients with aplastic anemia showed decreased levels of Tregs compared to healthy volunteers (1.31 ± 0.26 % vs 2.33 ± 0.36%, p=0.03, Figure 1A) . To further explore the mechanism involved in the increased expression of these two cell populations in AA patients, we examined the intracellular Ets-1 protein levels. AA patients showed statistically significant increased Ets-1 protein levels compared to controls (western blot, Figure 1B). Ets-1 protein levels correlated with the increased ABCs counts. Stat-1 protein levels were comparable between patients and controls, as previously described. In this study we explored the role of ABCs, the T-bet expressing B cells, in the immune pathogenesis of AA. We show for the first time that ABCs are increased along with increased pTfh cells, which are required for ABCs formation. The transcription factor Ets-1 is increased in AA patients and this could be one of the potential mechanisms for the increased ABCs. Whether these alterations are a consequence of increased IFN-g from polarized Th1 cells is not fully understood. One possible explanation could be that the IFN-g produced by activated T cells leads to increased numbers of circulating ABCs; these ABCs induce further the IFN-g production leading to a vicious cycle of IFN-g production in effect jeopardizing hematopoietic stem cell survival, leading to the clinical presentation of AA. Based on our results, it is proposed that T-bet has a central role not only in T but also in a small B cell subpopulation that seems central in AA pathogenesis. The development of agents that specifically target T-bet+ cells could possibly be used as novel therapeutic agents for AA but also for other autoimmune diseases. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.

The Ets protein Pointed P1 represses Asense expression in type II neuroblasts by activating Tailless.

Intermediate neural progenitors (INPs) boost the number and diversity of neurons generated from neural stem cells (NSCs) by undergoing transient proliferation. In the developing Drosophila brains, INPs are generated from type II neuroblasts (NBs). In order to maintain type II NB identity and their capability to produce INPs, the proneural protein Asense (Ase) needs to be silenced by the Ets transcription factor pointed P1 (PntP1), a master regulator of type II NB development. However, the molecular mechanisms underlying the PntP1-mediated suppression of Ase is still unclear. In this study, we utilized genetic and molecular approaches to determine the transcriptional property of PntP1 and identify the direct downstream effector of PntP1 and the cis-DNA elements that mediate the suppression of ase. Our results demonstrate that PntP1 directly activates the expression of the transcriptional repressor, Tailless (Tll), by binding to seven Ets-binding sites, and Tll in turn suppresses the expression of Ase in type II NBs by binding to two hexameric core half-site motifs. We further show that Tll provides positive feedback to maintain the expression of PntP1 and the identity of type II NBs. Thus, our study identifies a novel direct target of PntP1 and reveals mechanistic details of the specification and maintenance of the type II NB identity by PntP1.

HUWE1 Causes an Immune Imbalance in Immune Thrombocytopenic Purpura by Reducing the Number and Function of Treg Cells Through the Ubiquitination Degradation of Ets-1.

Background: Immune thrombocytopenic purpura (ITP) is an autoimmune bleeding disorder and the decreased number and immunosuppressive dysfunction of Treg cells are key promoters of ITP. However, their mechanisms in ITP development have not been fully clarified. Methods: HUWE1 mRNA and protein levels in CD4+ T cells in peripheral blood from ITP patients were assessed by quantitative real-time PCR and Western blot. HUWE1 function in ITP was estimated using flow cytometry, enzyme-linked immunosorbent assay and immunosuppression assay. Besides, the HUWE1 mechanism in reducing the number and function of Treg cells in ITP was investigated by immunoprecipitation, cycloheximide-chase assay, ubiquitin experiment and immunofluorescence assay. Results: HUWE1 expression was elevated in CD4+ T cells in peripheral blood from ITP patients and HUWE1 mRNA level was negatively correlated with platelet counts and Treg cell percentage. Moreover, the interference with HUWE1 increased the number of Treg cells and enhanced its immunosuppressive function, and the HUWE1 overexpression produced the opposite results. For the exploration of mechanism, HUWE1 interacted with E26 transformation-specific-1 (Ets-1) and this binding was dependent on the negative regulation of the phosphorylation level of Ets-1 (Thr38) and HUWE1 facilitated the ubiquitin degradation of Ets-1 protein to restrain Treg cell differentiation and weaken their immunosuppressive functions. The in vivo assay confirmed that the HUWE1 inhibitor alleviated ITP in mice. Conclusion: HUWE1 induced the immune imbalance in ITP by decreasing the number and weakening the function of Treg cells through the ubiquitination degradation of Ets-1.

ETS1, ELK1, and ETV4 Transcription Factors Regulate Angiopoietin-1 Signaling and the Angiogenic Response in Endothelial Cells.

BackgroundAngiopoietin-1 (Ang-1) is the main ligand of Tie-2 receptors. It promotes endothelial cell (EC) survival, migration, and differentiation. Little is known about the transcription factors (TFs) in ECs that are downstream from Tie-2 receptors.ObjectiveThe main objective of this study is to identify the roles of the ETS family of TFs in Ang-1 signaling and the angiogenic response.MethodsIn silico enrichment analyses that were designed to predict TF binding sites of the promotors of eighty-six Ang-1-upregulated genes showed significant enrichment of ETS1, ELK1, and ETV4 binding sites in ECs. Human umbilical vein endothelial cells (HUVECs) were exposed for different time periods to recombinant Ang-1 protein and mRNA levels of ETS1, ELK1, and ETV4 were measured with qPCR and intracellular localization of these transcription factors was assessed with immunofluorescence. Electrophoretic mobility shift assays and reporter assays were used to assess activation of ETS1, ELK1, and ETV4 in response to Ang-1 exposure. The functional roles of these TFs in Ang-1-induced endothelial cell survival, migration, differentiation, and gene regulation were evaluated by using a loss-of-function approach (transfection with siRNA oligos).ResultsAng-1 exposure increased ETS1 mRNA levels but had no effect on ELK1 or ETV4 levels. Immunostaining revealed that in control ECs, ETS1 has nuclear localization whereas ELK1 and ETV4 are localized to the nucleus and the cytosol. Ang-1 exposure increased nuclear intensity of ETS1 protein and enhanced nuclear mobilization of ELK1 and ETV4. Selective siRNA knockdown of ETS1, ELK1, and ETV4 showed that these TFs are required for Ang-1-induced EC survival and differentiation of cells, while ETS1 and ETV4 are required for Ang-1-induced EC migration. Moreover, ETS1, ELK1, and ETV4 knockdown inhibited Ang-1-induced upregulation of thirteen, eight, and nine pro-angiogenesis genes, respectively.ConclusionWe conclude that ETS1, ELK1, and ETV4 transcription factors play significant angiogenic roles in Ang-1 signaling in ECs.

Mechanism of tumor‑derived extracellular vesicles in regulating renal cell carcinoma progression by the delivery of MALAT1.

Renal cell carcinoma (RCC) is a major healthcare burden globally. Tumor-derived extracellular vesicles (EVs) contribute to the formation of a pro-metastatic microenvironment. In the present study, we explored the role and mechanism of RCC cell 786-O-derived EVs (786-O-EVs) in RCC. First, 786-O-EVs were extracted and identified, and EV internalization of RCC cells was observed. RCC cell malignant behaviors and long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) expression patterns were detected before and after 786-O-EV treatment. MALAT1 was intervened to evaluate RCC cell behaviors. The downstream mechanism involving MALAT1 was predicted. In addition, the relationship among MALAT1, transcription factor CP2 like 1 (TFCP2L1) and ETS proto-oncogene 1, transcription factor (ETS1) was analyzed. TFCP2L1 expression patterns were measured after 786-O-EV exposure. Tumor xenograft formation assay and lung metastasis model were adopted to verify the role of 786-O-EVs in vivo in RCC. It was found that 786-O-EVs could be internalized by RCC cells. 786-O-EVs promoted RCC cell malignant behaviors, accompanied by elevated MALAT1 expression levels. The 786-O-EVs with MALAT1 knockdown attenuated the promotive effect of sole 786-O-EVs on RCC cells. MALAT1 located ETS1 in the TFCP2L1 promoter and negatively regulated TFCP2L1, and ETS1 protein could specifically bind to MALAT1. 786-O-EVs enhanced the binding of ETS1 and the TFCP2L1 promoter and decreased TFCP2L1 expression. In vivo, 786-O-EVs promoted tumor growth and RCC lung metastasis, which was suppressed following inhibition of MALAT1. Our findings indicated that 786-O-EVs promoted RCC invasion and metastasis by transporting MALAT1 to promote the binding of transcription factor ETS1 and TFCP2L1 promoter.