Ets Family Research Articles

Abstract 1461: ETV6 deficiency and microsatellite enhancers drive transcriptional dysregulation in B-lymphoblastic leukemia

Abstract Subtypes of B-lymphoblastic leukemia (B-ALL) are defined by unique genetic aberrations and gene expression programs. ETV6-RUNX1+ (E-R+) B-ALL, a common pediatric subtype, shares a gene activation signature with “ETV6-RUNX1-like” B-ALL that lacks the E-R fusion gene. Both E-R+ and E-R-like B-ALL show frequent inactivating mutations or deletions of ETV6, which encodes an ETS family transcriptional repressor (core binding motif GGAA/T). Signature genes overexpressed in E-R+ B-ALL, such as EPOR and PIK3C3, were shown to sustain the malignant phenotype. We performed H3K27ac ChIP-Seq and ATAC-Seq on 26 B-cell cancer cell lines to identify subtype-specific active enhancer-like elements. Unbiased K-means clustering identified distal elements that were selectively acetylated in four E-R+ B-ALL cell lines (all with inactivation of the second ETV6 allele) and in a fifth B-ALL cell line with biallelic ETV6 deletion. Motif analysis of this cluster showed strong enrichment for tandem repeats of the sequence “GGAA” (p<10-100). Repeats with ≥6 tandem copies of “GGAA” were selectively acetylated and accessible in H3K27ac ChIP-Seq and ATAC-Seq datasets from primary patient samples of E-R+ B-ALL, but not from other genetic subtypes that lack ETV6 aberrations. Doxycycline-inducible expression of ETV6 in ETV6-deficient, E-R+ B-ALL cells led to significant growth inhibition. Restored ETV6 bound extensively to GGAA repeats (80% of ETV6 binding sites), repressed repeat enhancer acetylation, and repressed expression of adjacent E-R+ signature genes (n=40). Repression of a published E-R+ signature gene set was significant by GSEA analysis (p<0.001). Direct CRISPR-interference repression of 6 GGAA repeat enhancers led to significant downregulation of their associated E-R+ signature genes, including EPOR and PIK3C3. GGAA repeat enhancers are a known driver of Ewing sarcoma, where they are activated by oncogenic fusions of the ETS activators FLI1 or ERG. Active GGAA repeat enhancers and repeat-regulated genes in E-R+ B-ALL show only partial overlap with repeat-regulated genes in Ewings, possibly due to tissue-specific factors. Wild-type ERG and FLI1 are highly expressed in B-ALL. We found that knockdown of ERG, but not FLI1, led to significant downregulation of repeat enhancer-regulated E-R+ signature genes in B-ALL. ChIP-Seq showed that the ERG binds nearly all active GGAA repeat enhancers in three ETV6-deficient B-ALL cell lines, but not in ETV6-intact B-ALL. We identify GGAA repeat enhancers, sustained by ETV6 deficiency and strong ERG expression, as a unifying mechanism for a subtype-specific oncogenic gene expression program in B-ALL. Our findings may have crucial implications for E-R+ B-ALL modeling, as most GGAA repeats are not conserved between mice and humans. Mechanisms that activate leukemia-specific repeat enhancers may represent appealing targets for future therapeutic development. Citation Format: Joshua W. Goldman, Rohan Kodgule, Alexander C. Monovich, Travis Saari, Cody N. Hall, Niharika Rajesh, Juhi Gupta, Athalee Aguilar, Noah Brown, Mark Y. Chiang, Marcin P. Cieslik, Russell J. Ryan. ETV6 deficiency and microsatellite enhancers drive transcriptional dysregulation in B-lymphoblastic leukemia [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 1461.

Abstract 2210: The study of age and race genetic signatures of prostate cancer with formalin fixed paraffin embedded (FFPE) samples

Abstract Objectives: Diagnosis with prostate cancer (PCa) at younger age is a serious challenge in Military Health System. PCa onset at younger age is more likely to be aggressive in African American (AA) population. Also, application of formalin fixed paraffin embedded (FFPE) tissue in RNA based study was significantly hindered due to poor quality/quantity of RNA in archived pathology slides. This study is aimed to examine age/race associated gene signatures in military cohort with high representation of AA and younger population in FFPE samples for potential genetic mechanisms of health disparities and broaden tissue sample availability. Methods: A cohort of 41 Caucasian American (CA) and 20 AA non-familial PCa patients from the biospecimen bank of CPDR, WRNMMC were used. Specimens donated by radical prostatectomy patients who provided written consent and protocols approved by Institutional Review Boards. Total RNA was extracted from tumor and the adjacent normal epithelium of whole-mount FFPE tissue of matched specimens. Differential gene expression, in tumor versus normal tissues, between younger and older AA and CA patients were analyzed by NanoString using a customized CodeSet of 151 probes. The panel designed to detect 135 target transcripts, compiled from 34 genes implicated/associated with PCa progression, 27 PCa specific gene fusions, 25 cancer-associated genes, and 5 genes encoding ETS-family of transcription factors. The 27 over-expressed genes and 17 under-expressed genes in PCa were included as comparison to matched, benign epithelium. Additionally, 5 prostate stroma or epithelium specific genes were selected as control and 11 housekeeping genes were included for biological normalization. Results: The differentially expressed genes related to tumor-normal differences between AA older (66-73 years) and younger (42-58 years) as well as CA older and younger PCa patients with moderate Gleason score (3+3 and 3+4) sum were identified. After controlling for false discovery rates, panel of nine genes (ANXA2, C-MYC, VEGFR1, ERG, NPY, PSMA/FOLH1, TWIST1, MAOA, MYCN) uniquely associated with age/race were identified. Tumor vs normal ratios of ANXA2 and C-MYC expression in prostate tumors of older and younger patients were significantly enhanced. We found a clear association of VEGFR1 gene expression with CA young and c-MYC gene with AA young PCa. Conclusions: Using age/race as endpoint, we identified ANXA2, c-MYC and VEGFR1 as age and race associated PCa gene. Our findings highlight distinct racial and age-related differences and addresses the clinical utility of available genetic tests across old and young AA and CA men, which needs to be further validated in lager cohort. Further, our study also highlighted the utilization of FFPE tissue for NanoString assay and subsequent genetic signature analyses. Citation Format: Shashwat Sharad, Darryl Nousome, Gregory T. Chesnut, Hua Li. The study of age and race genetic signatures of prostate cancer with formalin fixed paraffin embedded (FFPE) samples [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 2210.

LncRNA 122049 suppresses apoptosis of renal tubular epithelial cells in ischemic AKI by targeting the miR-330-5p/ELK1 axis.

Several studies have reported that long non-coding RNAs (LncRNAs) were associated with the progression of acute kidney injury (AKI). However, the role and regulation mechanism of lncRNA122049 in ischemic AKI remains unknown. In the present study, we found that lncRNA 122049 protected against the ischemia/reperfusion (I/R) induced apoptosis in BUMPT cells. Mechanistically, the lncRNA 122049 directly sponged miR-330-5p, then increased the expression of ELK1(ETS transcription factor ELK1) to decrease renal cell apoptosis. In addition, miR-330-5p inhibitor completely reversed the pro-apoptotic effect of LncRNA 122049siRNA on I/R-induced BUMPT cells apoptosis. Finally, overexpression of lncRNA 122049 attenuated ischemic mice AKI via targeting of the miR-330-5p/ELK1 axis. Collectively, the data demonstrated that LncRNA 122049 prevented the I/R-induced renal cell apoptosis via regulation of the miR-330-5p/ELK1 axis, which brings new insights into the pathogenesis and potential targeted treatment of ischemic AKI.

E74 Like ETS Transcription Factor 3 is a Negative Regulator of Pathogenic Lamina Propria T Helper 17.1 Cells in Murine Colitis

ABSTRACT Interleukin-17A (IL-17A)-expressing T cells, including T helper 17 (Th17) and T helper 17.1 (Th17.1) cells, play a significant role in inflammatory bowel diseases (IBDs). Identifying the mechanisms underlying the heterogeneity and plasticity of IL-17A-expressing T cells is crucial for understanding and controlling their pathogenicity. The role of E74 like ETS transcription factor 3 (ELF3) in regulating the pathogenicity of IL-17A-expressing T cells has not been studied before. Dextran sulfate sodium was used to induce acute colitis in transgenic mice co-expressing IL-17A and enhanced green fluorescent protein (EGFP). IL-17A-expressing T cells were analyzed by flow cytometry. ELF3 expression was evaluated by reverse transcription and quantitative polymerase chain reaction. Lentivirus-mediated ELF3 overexpression was performed to assess the effect of ELF3 on Th17 and Th17.1 cells in vitro. The in vivo effect of ELF3 on Th17.1 cells was analyzed in an adoptive transfer colitis model. ELF3 was expressed by IL-17A-expressing T cells in the colonic lamina propria after colitis induction. Th17 cells and Th17.1 cells were distinguished based on the expression of C-X-C motif chemokine receptor 3, cytokine production, and key regulators. Th17 cells expressed higher ELF3 than Th17.1 cells. Ectopic ELF3 overexpression did not alter Th17 cell function while suppressing Th17.1 cell function in vitro. When adoptively transferred into Rag1 knockout mice to induce colitis, ELF3-overexpressing Th17.1 cells were less pathogenic than the control Th17.1 cells. ELF3 suppresses the pathogenicity of Th17.1 cells in colitis.

The Iron Deficiency-Regulated Small Protein Effector FEP3/IRON MAN1 Modulates Interaction of BRUTUS-LIKE1 With bHLH Subgroup IVc and POPEYE Transcription Factors.

In light of climate change and human population growth one of the most challenging tasks is to generate plants that are Fe-efficient, resilient to low Fe supply and Fe-biofortified. For such endeavors, it is crucial to understand the regulation of Fe acquisition and allocation in plants. One open question is how identified Fe-regulatory proteins comprising positive and negative regulators act together to steer Fe homeostasis. bHLH transcription factors (TFs) belonging to the subgroups IVb and IVc can initiate a bHLH cascade controlling the –Fe response in roots. In Arabidopsis thaliana, the –Fe-induced genes are sub-divided into several gene co-expression clusters controlled by different sets of TFs. Some of the co-expressed genes encode regulatory E3 ligase proteins BRUTUS (BTS)/BTS-LIKE (BTSL) and small proteins belonging to the group of FE UPTAKE-INDUCING PEPTIDE/IRON MAN (FEP/IMA). Recently, it was described that FEP1/IMA3 and FEP3/IMA1 proteins inhibit the repression of bHLH factors by BTS. We had postulated that –Fe-regulated co-expression clusters provide new information about regulatory protein interaction complexes. Here, we report a targeted yeast two-hybrid screen among 23 proteins of the –Fe response. This identified a novel protein interactome involving another E3 ligase, namely BTSL1, basic helix-loop-helix (bHLH) protein POPEYE (PYE) and transcription factors of the subgroup IVc as well as FEP3/IMA1. Because of the difficulty in stable BTSL1 protein expression in plant cells, we used a yeast two hybrid-based deletion mapping, homology modeling and molecular docking, to pinpoint interaction sites in BTSL1 and FEP3/IMA1. bHLH IVc TFs have similar residues at their C-terminus as FEP3/IMA1 interacting sites. FEP3/IMA1 attenuated interaction of BTSL1 and bHLH proteins in a yeast three-hybrid assay, in line with physiological data pointing to enhanced Fe acquisition and allocation in FEP3/IMA1 overexpression and btsl1 btsl2 mutant plants. Hence, exploiting –Fe-induced gene co-expression networks identified FEP3/IMA1 as a small effector protein that binds and inhibits the BTSL1 complex with PYE and bHLH subgroup IVc proteins. Structural analysis resolved interaction sites. This information helps improving models of Fe regulation and identifying novel targets for breeding of Fe-efficient crops.

Tissue-specific inhibition of protein sumoylation uncovers diverse SUMO functions during C. elegans vulval development.

The sumoylation (SUMO) pathway is involved in a variety of processes during C. elegans development, such as gonadal and vulval fate specification, cell cycle progression and maintenance of chromosome structure. The ubiquitous expression and pleiotropic effects have made it difficult to dissect the tissue-specific functions of the SUMO pathway and identify its target proteins. To overcome these challenges, we have established tools to block protein sumoylation and degrade sumoylated target proteins in a tissue-specific and temporally controlled manner. We employed the auxin-inducible protein degradation system (AID) to down-regulate the SUMO E3 ligase GEI-17 or the SUMO ortholog SMO-1, either in the vulval precursor cells (VPCs) or in the gonadal anchor cell (AC). Our results indicate that the SUMO pathway acts in multiple tissues to control different aspects of vulval development, such as AC positioning, basement membrane (BM) breaching, VPC fate specification and morphogenesis. Inhibition of protein sumoylation in the VPCs resulted in abnormal toroid formation and ectopic cell fusions during vulval morphogenesis. In particular, sumoylation of the ETS transcription factor LIN-1 at K169 is necessary for the proper contraction of the ventral vulA toroids. Thus, the SUMO pathway plays several distinct roles throughout vulval development.

The interaction between ETS transcription factor family members and microRNAs: A novel approach to cancer therapy.

In cancer biology, ETS transcription factors promote tumorigenesis by mediating transcriptional regulation of numerous genes via the conserved ETS DNA-binding domain. MicroRNAs (miRNAs) act as posttranscriptional regulators to regulate various tumor-promoting or tumor-suppressing factors. Interactions between ETS factors and miRNAs regulate complex tumor-promoting and tumor-suppressing networks. This review discusses the progress of ETS factors and miRNAs in cancer research in detail. We focused on characterizing the interaction of the miRNA/ETS axis with competing endogenous RNAs (ceRNAs) and its regulation in posttranslational modifications (PTMs) and the tumor microenvironment (TME). Finally, we explore the prospect of ETS factors and miRNAs in therapeutic intervention. Generally, interactions between ETS factors and miRNAs provide fresh perspectives into tumorigenesis and development and novel therapeutic approaches for malignant tumors.

3. Complex and cryptic EWSR1-FLI1 gene fusions and 1q jumping translocation in pediatric Ewing sarcomas

Ewing sarcomas are the second most frequent bone tumor of childhood that can also occur in soft tissue. They are highly aggressive with a survival of 30% for those with metastatic disease. They are genetically characterized by balanced reciprocal chromosomal translocations in which a member of the FET gene family is fused with an ETS transcription factor, with the most common translocation being the t(11;22)(q24;q12). The t(11;22) reciprocal translocation fuses the N-terminal transactivation domain of the Ewing sarcoma breakpoint region 1 protein (EWSR1) with the C-terminal DNA binding domain of the Friend leukemia integration 1 transcription factor (FLI1). The EWSR1-FLI1 fusion occurs in ∼90% of Ewing sarcoma cases and is a tumor-specific chimeric transcription factor that massively revamps the transcriptome. Therefore, the detection of EWSR1-FLI1 fusions is of important diagnostic value. Here, we present two pediatric Ewing sarcomas with atypical EWSR1-FLI1 fusions. The first case had a complex t(9;11;22)(q22;q24;q12) three-way translocation and a 1q jumping translocation by chromosome analysis. EWSR1 rearrangements using a EWSR1 break-apart FISH probe set revealed 5’ EWSR1 on der(22)(q12) and 3’ EWSR1 on der(9)(q22). Metaphase FISH using dual-color dual-fusion EWSR1-FLI1 FISH probe set revealed a EWSR1-FLI1 fusion at the derivative chromosome 22 involving the three-way translocation. 1q amplification by jumping translocations were confirmed by SNP microarray. The second case had a t(4;22)(q35;q12) reciprocal translocation and tetrasomy 8 by chromosome analysis. EWSR1 rearrangements using a EWSR1 break-apart FISH probe set revealed 5’ EWSR1 on der(22)(q12) and 3’ EWSR1 on der(4)(q35). Metaphase FISH using dual-color dual-fusion EWSR1-FLI1 FISH probe set revealed a EWSR1-FLI1 fusion at the derivative chromosome 22 involving the 4;22 translocation. Both cases had EWSR1-FLI1 gene fusions detected by Nanostring gene fusion panel. Recognizing complex and cryptic EWSR1-FLI1 gene fusions using various diagnostic methods is important for diagnosis, prognosis, and treatment outcome of pediatric Ewing sarcomas.

FGF/MAPK/Ets signaling in Xenopus ectoderm contributes to neural induction and patterning in an autonomous and paracrine manner, respectively.

FGF and anti-BMP signals from the Spemann organizer of mesodermal origin are essential for Xenopus neural development from gastrula ectoderm. However, the detailed cellular and molecular mechanisms of signaling, especially those underlying the neural induction process, are still controversial. We show here that the expression of early neural marker genes such as sox2 and otx2 is suppressed both in vivo and in vitro, when ectoderm cells are loaded with a dominant-negative construct of Ets transcription factors or a translation-blocking antisense FGF2 MO or FGF8 MO, respectively. This indicates that the expression of these FGF signaling molecules in ectoderm cells contributes significantly to neural induction, in contrast to the "neural default model" that has been emphasized, in which anti-BMP signaling from the organizer plays a major role in the neural induction in Xenopus. Our results indicate that cell-autonomous FGF signaling between ectoderm cells, rather than paracrine signaling from organizer cells, directly induces the expression of sox2 and otx2 via the FGF2, FGF8/MAPK/Ets pathway at very low signaling levels. This is independent of inhibiting BMP signaling via the FGF/MAPK/Smad pathway, which has been proposed as the primary pathway for FGF signaling in Xenopus neural induction. Using cultured ectoderm cells, we also obtained results suggesting that the mode of contribution of FGF signaling to neural development is altered during the subsequent neural patterning stage. As we increase the amounts of FGF in the culture medium, anterior neural genes activated at low FGF signaling levels are suppressed, and instead position-specific, more posterior neural genes are activated in a dose-dependent manner via the FGF/MAPK/Ets pathway. These results support the claim that morphogenic FGFs from the organizer diffuse in a paracrine manner through the plane of the induced neuroectoderm, causing anteroposterior patterning of neural tissue.

Protein Tyrosine Phosphatase SHP2 Controls Interleukin-8 Expression in Breast Cancer Cells

Treatment of metastasis remains a clinical challenge and the majority of breast cancer-related deaths are the result of drug-resistant metastases. The protein tyrosine phosphatase SHP2 encoded by the proto-oncogene PTPN11 promotes breast cancer progression. Inhibition of SHP2 has been shown to decrease metastases formation in various breast cancer models, but specific downstream effectors of SHP2 remain poorly characterized. Certain cytokines in the metastatic cascade facilitate local invasion and promote metastatic colonization. In this study, we investigated cytokines affected by SHP2 that could be relevant for its pro-tumorigenic properties. We used a cytokine array to investigate differentially released cytokines in the supernatant of SHP2 inhibitor-treated breast cancer cells. Expression of CXCL8 transcripts and protein abundance were assessed in human breast cancer cell lines in which we blocked SHP2 using shRNA constructs or an allosteric inhibitor. The impact of SHP2 inhibition on the phospho-tyrosine-proteome and signaling was determined using mass spectrometry. From previously published RNAseq data (Aceto et al. in Nat. Med. 18:529–37, 2012), we computed transcription factor activities using an integrated system for motif activity response analysis (ISMARA) (Balwierz et al. in Genome Res. 24:869–84, 2014). Finally, using siRNA against ETS1, we investigated whether ETS1 directly influences CXCL8 expression levels. We found that IL-8 is one of the most downregulated cytokines in cell supernatants upon SHP2 blockade, with a twofold decrease in CXCL8 transcripts and a fourfold decrease in IL-8 protein. These effects were also observed in preclinical tumor models. Analysis of the phospho-tyrosine-proteome revealed that several effectors of the mitogen-activated protein kinase (MAPK) pathway are downregulated upon SHP2 inhibition in vitro. MEK1/2 inhibition consistently reduced IL-8 levels in breast cancer cell supernatants. Computational analysis of RNAseq data from SHP2-depleted tumors revealed reduced activity of the transcription factor ETS1, a direct target of ERK and a transcription factor reported to regulate IL-8 expression. Our work reveals that SHP2 mediates breast cancer progression by enhancing the production and secretion of the pro-metastatic cytokine IL-8. We also provide mechanistic insights into the effects of SHP2 inhibition and its downstream repercussions. Overall, these results support a rationale for targeting SHP2 in breast cancer.

Constraint-Based, Score-Based and Hybrid Algorithms to Construct Bayesian Gene Networks in the Bovine Transcriptome.

Simple SummaryIn this study, we investigated and compared six different Bayesian network algorithms from three different categories to identify hub genes critical to gene expression networks activated in response to progesterone in the bovine uterus. We observed many common hub genes identified between constraint-based algorithms (CBAs) and hybrid algorithms (HAs), while it appeared that score-based algorithm (SBA) methods led to more accurate and relevant predictions of core genes. The results revealed that the identification of hub genes was affected by the type of network reconstruction and by the subsequently used topological parameters. Two identified genes known to have roles during pregnancy are ISG15 and DGAT2. The identified hub genes are associated with biological processes such as amino acid metabolism, hormonal signaling pathways and the immune system. Our analysis revealed a role for miRNAs in the regulation of this system. The biological and physiological roles (enzymatic and hormonal effects) of unannotated identified hub genes should be functionally validated by further studies.Bayesian gene networks are powerful for modelling causal relationships and incorporating prior knowledge for making inferences about relationships. We used three algorithms to construct Bayesian gene networks around genes expressed in the bovine uterus and compared the efficacies of the algorithms. Dataset GSE33030 from the Gene Expression Omnibus (GEO) repository was analyzed using different algorithms for hub gene expression due to the effect of progesterone on bovine endometrial tissue following conception. Six different algorithms (grow-shrink, max-min parent children, tabu search, hill-climbing, max-min hill-climbing and restricted maximum) were compared in three higher categories, including constraint-based, score-based and hybrid algorithms. Gene network parameters were estimated using the bnlearn bundle, which is a Bayesian network structure learning toolbox implemented in R. The results obtained indicated the tabu search algorithm identified the highest degree between genes (390), Markov blankets (25.64), neighborhood sizes (8.76) and branching factors (4.38). The results showed that the highest number of shared hub genes (e.g., proline dehydrogenase 1 (PRODH), Sam-pointed domain containing Ets transcription factor (SPDEF), monocyte-to-macrophage differentiation associated 2 (MMD2), semaphorin 3E (SEMA3E), solute carrier family 27 member 6 (SLC27A6) and actin gamma 2 (ACTG2)) was seen between the hybrid and the constraint-based algorithms, and these genes could be recommended as central to the GSE33030 data series. Functional annotation of the hub genes in uterine tissue during progesterone treatment in the pregnancy period showed that the predicted hub genes were involved in extracellular pathways, lipid and protein metabolism, protein structure and post-translational processes. The identified hub genes obtained by the score-based algorithms had a role in 2-arachidonoylglycerol and enzyme modulation. In conclusion, different algorithms and subsequent topological parameters were used to identify hub genes to better illuminate pathways acting in response to progesterone treatment in the bovine uterus, which should help with our understanding of gene regulatory networks in complex trait expression.

GABPA-activated TGFBR2 transcription inhibits aggressiveness but is epigenetically erased by oncometabolites in renal cell carcinoma

BackgroundThe ETS transcription factor GABPA has long been thought of as an oncogenic factor and recently suggested as a target for cancer therapy due to its critical effect on telomerase activation, but the role of GABPA in clear cell renal cell carcinoma (ccRCC) is unclear. In addition, ccRCC is characterized by metabolic reprograming with aberrant accumulation of L-2-hydroxyglurate (L-2HG), an oncometabolite that has been shown to promote ccRCC development and progression by inducing DNA methylation, however, its downstream effectors remain poorly defined.MethodssiRNAs and expression vectors were used to manipulate the expression of GABPA and other factors and to determine cellular/molecular and phenotypic alterations. RNA sequencing and ChIP assays were performed to identify GABPA target genes. A human ccRCC xenograft model in mice was used to evaluate the effect of GABPA overexpression on in vivo tumorigenesis and metastasis. ccRCC cells were incubated with L-2-HG to analyze GABPA expression and methylation. We carried out immunohistochemistry on patient specimens and TCGA dataset analyses to assess the effect of GABPA on ccRCC survival.ResultsGABPA depletion, although inhibiting telomerase expression, robustly enhanced proliferation, invasion and stemness of ccRCC cells, whereas GABPA overexpression exhibited opposite effects, strongly inhibiting in vivo metastasis and carcinogenesis. TGFBR2 was identified as the GABPA target gene through which GABPA governed the TGFβ signaling to dictate ccRCC phenotypes. GABPA and TGFBR2 phenocopies each other in ccRCC cells. Higher GABPA or TGFBR2 expression predicted longer survival in patients with ccRCC. Incubation of ccRCC cells with L-2-HG mimics GABPA-knockdown-mediated phenotypic alterations. L-2-HG silenced the expression of GABPA in ccRCC cells by increasing its methylation.ConclusionsGABPA acts as a tumor suppressor by stimulating TGFBR2 expression and TGFβ signaling, while L-2-HG epigenetically inhibits GABPA expression, disrupting the GABPA-TGFβ loop to drive ccRCC aggressiveness. These results exemplify how oncometabolites erase tumor suppressive function for cancer development/progression. Restoring GABPA expression using DNA methylation inhibitors or other approaches, rather than targeting it, may be a novel strategy for ccRCC therapy.

Upregulation of the Mevalonate Pathway through EWSR1-FLI1/EGR2 Regulatory Axis Confers Ewing Cells Exquisite Sensitivity to Statins.

Simple SummaryThe objective of this project was to search for new dependencies in Ewing sarcoma, a deadly disease for which new therapeutic approaches are urgently needed. A pharmacological screening of off-patent approved drugs (FDA agency) and the investigation of downstream targets of EGR2 were performed. The two approaches showed the MVA pathway as a major dependency in Ewing sarcoma and statin, an inhibitor of this pathway, as a potential new therapeutic agent for the treatment of Ewing sarcoma.Ewing sarcoma (EwS) is an aggressive primary bone cancer in children and young adults characterized by oncogenic fusions between genes encoding FET-RNA-binding proteins and ETS transcription factors, the most frequent fusion being EWSR1-FLI1. We show that EGR2, an Ewing-susceptibility gene and an essential direct target of EWSR1-FLI1, directly regulates the transcription of genes encoding key enzymes of the mevalonate (MVA) pathway. Consequently, Ewing sarcoma is one of the tumors that expresses the highest levels of mevalonate pathway genes. Moreover, genome-wide screens indicate that MVA pathway genes constitute major dependencies of Ewing cells. Accordingly, the statin inhibitors of HMG-CoA-reductase, a rate-limiting enzyme of the MVA pathway, demonstrate cytotoxicity in EwS. Statins induce increased ROS and lipid peroxidation levels, as well as decreased membrane localization of prenylated proteins, such as small GTP proteins. These metabolic effects lead to an alteration in the dynamics of S-phase progression and to apoptosis. Statin-induced effects can be rescued by downstream products of the MVA pathway. Finally, we further show that statins impair tumor growth in different Ewing PDX models. Altogether, the data show that statins, which are off-patent, well-tolerated, and inexpensive compounds, should be strongly considered in the therapeutic arsenal against this deadly childhood disease.

ETS transcription factor ELF3 (ESE-1) is a cell cycle regulator in benign and malignant prostate.

This study aimed to elucidate the role of ELF3, an ETS family member in normal prostate growth and prostate cancer. Silencing ELF3 in both benign prostate (BPH‐1) and prostate cancer (PC3) cell lines resulted in decreased colony‐forming ability, inhibition of cell migration and reduced cell viability due to cell cycle arrest, establishing ELF3 as a cell cycle regulator. Increased ELF3 expression in more advanced prostate tumours was shown by immunostaining of tissue microarrays and from analysis of gene expression and genetic alteration studies. This study indicates that ELF3 functions not only as a part of normal prostate epithelial growth but also as a potential oncogene in advanced prostate cancers.

Knocking down ETS Proto-oncogene 1 (ETS1) alleviates the pyroptosis of renal tubular epithelial cells in patients with acute kidney injury by regulating the NLR family pyrin domain containing 3 (NLRP3) transcription

ABSTRACT Acute kidney injury (AKI) has a high mortality rate, but its pathogenesis remains unclear Lipopolysaccharide (LPS)-mediated renal tubular epithelial pyroptosis is involved in the pathogenesis of AKI. NLR family of pyrin domains containing 3 (NLRP3) plays an important role in pyroptosis. To further understand the transcriptional regulation mechanism of NLRP3, the peripheral blood of patients with AKI was analyzed in this study, showing that the levels of NLRP3 and cell pyroptosis in patients with AKI were significantly higher than those in normal controls. Furthermore, elevated levels of NLRP3 and cell pyroptosis were found in renal tubular epithelial cells after LPS treatment. Transcription factor ETS Proto-Oncogene 1 (ETS1) could bind to the upstream promoter transcription site of NLRP3 to transactivate NLRP3 in renal tubular epithelial cells. The cell pyroptosis level also decreased by knocking down ETS1. It is concluded that knocking down of ETS1 may reduce the renal tubular epithelial pyroptosis by regulating the transcription of NLRP3, thus relieving AKI. ETS1 is expected to be a molecular target for the treatment of AKI.

Differential Etv2 threshold requirement for endothelial and erythropoietic development.

SUMMARYEndothelial and erythropoietic lineages arise from a common developmental progenitor. Etv2 is a master transcriptional regulator required for the development of both lineages. However, the mechanisms through which Etv2 initiates the gene-regulatory networks (GRNs) for endothelial and erythropoietic specification and how the two GRNs diverge downstream of Etv2 remain incompletely understood. Here, by analyzing a hypomorphic Etv2 mutant, we demonstrate different threshold requirements for initiation of the downstream GRNs for endothelial and erythropoietic development. We show that Etv2 functions directly in a coherent feedforward transcriptional network for vascular endothelial development, and a low level of Etv2 expression is sufficient to induce and sustain the endothelial GRN. In contrast, Etv2 induces the erythropoietic GRN indirectly via activation of Tal1, which requires a significantly higher threshold of Etv2 to initiate and sustain erythropoietic development. These results provide important mechanistic insight into the divergence of the endothelial and erythropoietic lineages.

A Transcriptional Regulation Bioinformatics Pipeline to Predict Co‐Regulated Genes in Vascular Smooth Muscle Cell Phenotypic Transitions During Atherosclerosis

The rupture of vulnerable or advanced atherosclerotic lesions contributes to myocardial infarctions and strokes, the leading causes of death globally. During all stages of atherosclerosis, vascular smooth muscle cells (VSMCs) are recruited as a major source of plaque cells within lesions, where they undergo phenotypic modulation and give rise to both plaque‐stabilizing and destabilizing phenotypes. Because most atherosclerosis studies test the functional role of single transcription factors, genes, and proteins in VSMCs, many aspects of how VSMC plasticity can be clinically exploited to prevent the rupture of vulnerable atherosclerotic lesions remain unclear. Systems biology approaches have emerged as a promising avenue for characterizing cellular dynamics at the organismal level, but they remain under‐utilized in the field of cardiovascular medicine. This study proposes a novel systems biology pipeline that integrates experimental and computational tools with large datasets measuring the transcriptomes and epigenomes of phenotypically transitioning VSMCs. Our pipeline allows users to identify candidate transcription factors that co‐regulate mouse or human gene sets controlling specific VSMC phenotypic transitions during atherosclerosis. The computational algorithms developed in this workflow integrate an array of publicly available databases, such as the Ensembl genome database and the JASPAR transcription factor database. To demonstrate the translational potential of our pipeline, we used it to test the hypothesis that the murine mesenchymal marker stem cell antigen‐1 Sca1(Ly6a) has a human ortholog, which has not been previously validated as a target in clinical therapies. To test the hypothesis that there are sets of genes co‐regulated with Ly6ain human VSMCs which, when expressed in concert, promote a distinct phenotypic state marked by Ly6ain mice, we integrated genomic and ATAC‐seq analyses of VSMCs to identify murine genes with human orthologs that bind Ly6a‐specific transcription factors. Specifically, we predict that the transcription factors ETS1, KLF5, GATA6, and SOX10 co‐regulate sets of human genes with Ly6a. While prior studies have associated these transcription factors with VSMC proliferation and phenotypic modulation, we specifically predict these Ly6a‐specific transcription factor co‐regulate the following human gene sets: PHACTR1, EFHC1, LAMC2(ETS1); TTC33, RUNX1, KIAA1217(KLF5); TMEM50A(GATA6); and ZBTB20, MYLK2(SOX10). To validate these predictions, we will determine if these transcription factors and their co‐regulated genes are expressed in single cell RNA‐sequencing analyses of human carotid arteries and can be detected with immunofluorescence staining of human coronary artery sections. We will also use siRNA to target these candidate transcription factors and genes in cultured VSMCs. The results of our pipeline will help identify novel transcriptional regulatory networks in VSMCs that can be therapeutically targeted for the treatment of advanced atherosclerosis.

Immune-Related Biomarkers Associated with Lung Metastasis from the Colorectal Cancer Microenvironment.

Immune-associated biomarkers can predict lung metastases from colorectal cancer. Differentially expressed genes (DEGs) were screened from sample data extracted from gene expression omnibus (GEO) database. The DEGs were screened from the lung metastasis (LM) and primary cancer (PC) groups of the Moffitt Cancer Center cohort dataset. Then, the tumor immune microenvironment and abundance of immune cell infiltration analyses were performed, and the immune-related DEGs were retrieved. In addition, the transcription factor (TF)-miRNA-mRNA network was constructed and enrichment analyses of the immune-related DEGs and upregulated and downregulated DEGs were carried out. Then, the protein-protein interaction (PPI) network was conducted and the drug-gene interaction was predicted. A total of 268 DEGs were screened. The Immune_Score of samples in the LM group was significantly higher compared with the PC group. The infiltration ratio of M0 macrophages and M2 macrophages of samples was higher than others. A total of 54 immune-related DEGs in M0 macrophages were screened. Moreover, the TF-miRNA-mRNA network was constructed among 8 miRNA-mRNA and 50 TF-mRNA, and the secreted phosphoprotein 1 was regulated by 12 TFs, and the oxidized low-density lipoprotein receptor 1 was regulated by 3 miRNAs and 3 TFs. The TF SAM pointed domain containing ETS TF was also a downregulated DEG. The Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the DEGs in the TF-miRNA-mRNA network were mainly involved in the interleukin-7 signaling pathway and cell adhesion molecules. In total, 23 protein interactions in this PPI network of M0 macrophage cells were involved in 27 mRNAs. There were 38 drug-gene interactions of immune-related DEGs of M0 macrophage cells predicted to contain 34 small molecule drugs and 8 mRNAs. Finally, the CON cohort dataset verified that the infiltration ratio of M0 and M2 macrophages of the samples was higher.

Structural and functional analysis of cancer related proteins: A critical target for drug development

Protein kinases are involved in cancer metabolism and have been targeted for structure‐based drug development. The crystals structures of several clinically important protein kinase‐inhibitor complexes have been determined, which provide the mechanism of inhibition of protein kinases. In our laboratory, we are working on structure and function analysis on two protein kinases e.g. (i) a non‐receptor tyrosine kinase expressed in hepatocellular carcinoma (TEC) and Class I Phosphoinositide 3‐Kinase (PI3K). We have cloned and expressed both kinases in E. coli and looking forward for biochemical and structural characterization of both proteins.In addition, we are also working on structural and biochemical characterization of Ergp55 protein belongs to Ets family of transcription factor. The Ergp55 is involved in various development processes and regulation of cancer metabolism. Using biochemical assay, we have identified the part of Ergp55 protein that mediates the auto‐inhibition using E74 and cfos promoter DNA sequences. We have also crystallized and solved the crystal structure of ETS domain of Ergp55 in native and its complexes with E74 and cFOS promoter DNA sequences. The structure analysis provides the insights into DNA sequence discrimination by ETS domain of Ergp55 protein. These studies will aid in designing the compounds, which stabilize the inhibited form of Ergp55 and inhibits its binding to DNA. Based on crystal structures, we have designed the DNA binding compounds, which interfere in ETS‐DNA binding and proved to be potential drugs against prostate cancer.

Role of transcription factor RUNX1 in the maintenance of T helper cell epigenome and rapid recall response

Abstract Memory T cells exhibit an accelerated response after a second encounter with the same antigen. In our previous studies, we showed that the ability to produce effector molecules within minutes of the secondary exposure to antigen (“rapid recall response”) is associated with the epigenetic state of the rapid recall genes. This memory epigenome is established during the primary T cell encounter with its cognate antigen. To maintain a “rapid recall response” even when antigen is eliminated, resting memory T cells would have to maintain open chromatin signature for years despite episodic homeostatic T cell proliferation. However, the mechanisms underlying the long-term maintenance of this epigenetic pattern remains unclear. Here, we used 10X Multiome (scRNA-seq and scATAC-seq obtained from the same cells) analysis to show that the association between the chromatin state of memory cells and their cytokine expression upon activation is Th lineage specific. Bioinformatic analysis showed that open chromatin specific for memory but not naïve CD4 T cells is enriched with motifs of constitutively expressed transcription factors RUNX, SP1, and ETS. Our preliminary Cut&Tag data in total human resting CD4 T cells demonstrates that RUNX1 binds DNA close to T helper cytokines genes (IL3, IL4, IL9, IL10, IL13, IFNG) at “memory-specific” ATAC-seq regions, suggesting the role of RUNX1 in maintaining this “rapid recall” epigenome. Together with the dynamic model of transcriptome and epigenome rearrangements this data could help us to understand how memory T cells establish a platform that enables and maintains their unique ability to respond more rapidly to subsequent encounters with antigen. Supported by R01AI153442-01A1 from NIAID, NIH