Academic literature on the topic 'ETS2-ERG'

Abstract Abstract 1600 Poster Board I-626 Background The V-ets erythroblastosis virus E26 (ETS) oncogene family is one of the largest families of transcription factors. ETS transcription factors are characterized by two major functional domains, a transcription domain and an evolutionarily highly conserved DNA-binding domain, also known as ETS domain that mediates binding to purine-rich DNA sequences. Most ETS family proteins are nuclear targets for activation of the Ras-MAP kinase signalling pathway. Therefore, they play a significant role in regulating cellular functions such as cell growth, apoptosis, development and differentiation. ETS transcription factors have been implicated in leukemia by chromosomal rearrangement, and more commonly by gene amplification and/or overexpression. Moreover, overexpression of ERG was shown to be an adverse predictor for clinical outcome in AML with normal cytogenetics (CN). In our recent study on complex karyotype AML, array-CGH (comparative genomic hybridization) analysis identified small genomic amplifications affecting ERG/ETS2 in 21q22 and ETS1/FLI1 in 11q23 in about 10% of the cases. Correlation with global gene expression profiling showed that ERG and ETS2 as well as ETS1 and FLI1 were overexpressed in these cases. Aims: To evaluate expression levels of ERG, ETS2, ETS1 and FLI1 in a large cohort of younger (16 to 60 years of age) adult CN-AML patients (pts) and their impact on clinical outcome. Methods The expression of ERG, ETS2, ETS1 and FLI1 was determined by quantitative real-time reverse transcriptase polymerase chain reaction (qPCR) assay in 343 CN-AML pts who were entered on 3 AMLSG treatment protocols (AMLHD93, AML HD98-A, AMLSG 07-04). ERG, ETS2, ETS1, and FLI1 were dichotomized into two major groups according to their expression levels. The upper quartile was chosen as the cut point and the set of patients with gene expression above were defined as Q4 group. Univariable as well as multivariable regression models were used to evaluate the influence of ERG, ETS2, ETS1 and FLI1 on induction success, event-free, relapse-free and overall survival. Multivariable analyses were stratified for AMLSG treatment protocols. Results Partial correlation analysis revealed positive correlations of expression levels between ETS2 and ERG (ρ=0.45) being the strongest, followed by ERG and FLI1 (ρ=0.4), as well as ETS1 and FLI1 (ρ=0.31). Correlation of ERG, ETS2, ETS1 and FLI1 with white blood count (WBC) revealed a significant association between high gene expression (Q4) and elevated WBC (ERG, p=0.004; ETS2, p=0.002, FLI1 p<0.001), whereas high expression of ETS1 was associated with a significantly lower WBC (p<0.001). Univariable as well as multivariable analyses on induction success revealed high ETS2 as an unfavourable marker (OR, 0.29, p=0.01). In univariable analysis, there was a significantly inferior relapse-free survival (RFS) and overall survival (OS) for high ERG (p=.01; p=.06, respectively) and high ETS2 (p=.002; p=.03, respectively) that was even more pronounced when both ERG Q4 and ETS2 Q4 (ERG Q4 ∩ ETS2 Q4) (p<0.001; p=.001, respectively) were included as one variable and compared with the rest. In multivariable analysis for the endpoints event-free survival (EFS), RFS and OS, a significant effect was found for RFS for ERG Q4 ∩ ETS2 Q4 (p=.002); the only significant variables that consistently appeared in the model were NPM1mut, FLT3-ITDpos and WBC. In subgroup analysis for the genotypes CEBPAmut, NPM1mut/FLT3-ITDneg, and all others (NPM1mut/FLT3-ITDpos, NPM1wt/FLT3-ITDpos, NPM1wt/FLT3-ITDneg) according to the hierarchical model, ERG Q4 was associated with an inferior EFS (p=.04) and OS (p=.03) in the favorable CEBPAmut genotype and became even more significant for the variable ERG Q4 ∩ ETS2 Q4 (EFS, p=.007, RFS, p=.002; OS, p=.06, respectively). For the NPM1mut/FLT3-ITDneg subgroup, again ERG Q4 ∩ ETS2 Q4 was associated with an adverse RFS (p=.04), but not with OS (p=0.07). Conclusions In our study on a large cohort of homogenously treated CN-AML patients, ERG and ETS2 expression were highly correlated. Overexpression of both genes had a significant impact on clinical outcome of CN-AML patients. Moreover, adverse effects of high ERG and high ETS2 expression on prognosis were also shown for the genetic AML subgroups CEBPAmut and NPM1mut/FLT3-ITDneg. Disclosures No relevant conflicts of interest to declare.

Abstract Specific genes that contribute to Transient Myeloproliferative Disorder (TMD) and DS Acute Megakaryocytic Leukemia (AMKL) remain largely undefined. To date, only the X-linked transcription factor GATA1, which is mutated in these disorders, has been implicated in contributing to DS hematopoietic malignancies. We recently reported that overexpression of either of the chromosome 21 ETS family members ERG or ETS2 led to a dramatic increase in megakaryopoeisis from wild-type or Gata1-knockdown fetal liver hematopoietic progenitors. Furthermore, we demonstrated that expression of ETS2 or ERG caused a significant increase in megakaryocyte colony formation and led to immortalization of Gata1-knockdown fetal liver hematopoietic progenitors in vitro. In new studies to define the mechanisms by which these ETS proteins promote megakaryopoiesis, we discovered that the ETS family member FLI1, which shares significant homology with ERG, significantly increased CD41 and CD42 expression and the degree of polyploidization of megakaryocytes in vitro in a manner that was nearly identical to that of ectopic ERG. Furthermore, FLI1 also immortalized Gata1-knockdown fetal liver progenitors. To characterize the effects of ectopic ETS family members on transcription, we performed genome wide expression profiling using RNA isolated from fourth generation immortalized Gata1-knockdown cells expressing ETS2, ERG, FLI1 or GFP alone. Principal component analysis showed that the ERG and FLI1 gene expression patterns clustered tightly with one another and were well separated from both the MIGR1 and ETS2 signatures. Unsupervised hierarchical clustering further revealed that 726 genes were differentially expressed in FLI1, ERG, ETS2 immortalized cells compared to MIGR1 control cells. Of these, genes associated with the erythroid lineage, including Klf1, Lmo2, glycophorin A, Egr1, Ank1 and Eraf were strongly repressed. These findings are consistent with recent publications showing that FLI1 promotes the development of megakaryocytes at the expense of red blood cells and suggest that ERG and ETS2 mimic FLI1 in our assays. Notably, we did not observe significant increases in the expression of Hox genes or Meis1, genes whose expression is often associated with transformed hematopoietic cells. However, we did detect increased expression of Bmi1, which has an essential role in regulating the proliferative capacity of both normal and malignant hematopoietic progenitors, as well as increased expression of Jak2 and Stat5, two genes whose activation is associated with myeloproliferative disease and megakaryocytic transformation, in ETS-family transduced cells. Next, to determine whether ETS-family expression affected the immunophenotype of Gata1-knockdown fetal liver progenitors, immortalized cells were assessed for cell surface expression of c-kit and CD41. ERG and FLI1 expressing colonies included a substantial proportion of CD41+/c-kit- cells, indicative of megakaryocytic maturation. Furthermore, colonies formed from progenitors transduced with ERG were comprised of greater than 90% CD41+ cells, confirming that nearly all of the cells within the immortalized colonies are of the megakaryocyte lineage. Finally, because our gene array data showed increased Jak2 and Stat5 mRNA levels in ETS-family transduced cells, and activating mutations in JAK3 are associated with AMKL, we assessed whether JAK/STAT signaling was altered in the immortalized cells. Using flow cytometry, we found that cells within the ERG and FLI1 cultures consistently showed significant increases in phospho-STAT3 staining in comparison to the MIGR1 control. The increases in STAT3 phosphorylation suggest that ERG and FLI1 promote immortalization of Gata1-knockdown fetal liver progenitors in part by activation of the JAK/STAT pathway. Together our data support the hypothesis that an increased dosage of ETS factors, including ETS2 and ERG, contribute to a pro-megakaryocytic phenotype and to the leukemogenic process in DS TMD and AMKL.

Abstract ETS2 and ERG are transcription factors, encoded on human chromosome 21 (Hsa21), that have been implicated in human cancer. People with Down syndrome (DS), who are trisomic for Hsa21, are predisposed to acute megakaryoblastic leukemia (AMKL). DS-AMKL blasts harbor a mutation in GATA1, which leads to loss of full-length protein but expression of the GATA-1s isoform. To assess the consequences of ETS protein misexpression on megakaryopoiesis, we expressed ETS2, ERG, and the related protein FLI-1 in wild-type and Gata1 mutant murine fetal liver progenitors. These studies revealed that ETS2, ERG, and FLI-1 facilitated the expansion of megakaryocytes from wild-type, Gata1-knockdown, and Gata1s knockin progenitors, but none of the genes could overcome the differentiation block characteristic of the Gata1-knockdown megakaryocytes. Although overexpression of ETS proteins increased the proportion of CD41+ cells generated from Gata1s-knockin progenitors, their expression led to a significant reduction in the more mature CD42 fraction. Serial replating assays revealed that overexpression of ERG or FLI-1 immortalized Gata1-knockdown and Gata1s knockin, but not wild-type, fetal liver progenitors. Immortalization was accompanied by activation of the JAK/STAT pathway, commonly seen in megakaryocytic malignancies. These findings provide evidence for synergy between alterations in GATA-1 and overexpression of ETS proteins in aberrant megakaryopoiesis.

Abstract ETS2 and ERG are two ETS family transcription factors encoded on chromosome 21 that have been implicated in leukemia and other types of cancer. In the setting of trisomy 21 (e.g. in individuals with Down syndrome) there is a significant increase in the propensity for leukemia, including AMKL (acute megakaryoblastic leukemia). It has been theorized that genes located on chromosome 21 may contribute to the predisposition of children with Down syndrome for pediatric AMKL, possibly by collaborating with mutations in the transcriptional regulator GATA1. To test the oncogenic potential of these genes in the hematopoietic system, ETS2 and the ERG isoform, ERG1, were over-expressed in both wild-type and GATA1 knockdown (ΔneoΔHS) fetal liver hematopoietic progenitors and the effects on growth and differentiation were studied. We discovered that over-expression of either ETS2 or ERG1 in wild-type progenitors increased the formation of CD41 positive megakaryocytes by 2–3 fold in liquid culture. However, whereas ETS2 appeared to primarily promote the early stages of megakaryopoiesis, ERG1 over-expression led to a dramatic rise in the percentage of cells expressing CD42, a marker of more mature megakaryocytes. Ectopic ERG1 expression also affected the ploidy profile, with nearly all cells containing ≥4N DNA content. Quantitative real time RT-PCR analysis of lineage specific genes confirmed that expression of megakaryocyte genes, including GPIIb (CD41), platelet factor 4 (PF4), and ß1-tubulin, was significantly increased in undifferentiated cells over-expressing either ETS2 or ERG1. These results show that over-expression of ETS2 or ERG1 rapidly initiates the megakaryocyte program. Over-expression of either ETS2 or ERG1 in GATA1 knockdown fetal liver cells similarly led to a 2.5–5 fold increase in the formation of CD41 positive megakaryocytes in liquid culture; however, neither ETS2 or ERG1 could circumvent the differentiation block that is characteristic of the GATA1 knockdown strain, including the inability to form proplatelet extensions. We next assessed the capacity of ETS2 or ERG1 over-expression to alter the colony forming ability of wild-type and GATA1 knockdown fetal liver progenitors. Both ETS2 and ERG1 expression led to a significant increase in CFU-MK derived from wild-type progenitors, as evidenced by an increase in the number of acetylcholinesterase positive colonies. In contrast, neither gene altered the formation of CFU-MK when over-expressed in the GATA1 knockdown progenitors, which already show a prominent enhancement in megakaryocyte colony formation over wild-type fetal liver cells. Finally, serial re-plating assays performed with GATA1 knockdown fetal liver cells showed that ectopic expression of either ETS2 or ERG1 could immortalize hematopoietic progenitors, whereas only ERG1 could enhance colony re-plating of wild-type cells. These findings provide evidence for a specific synergy between GATA-1 deficiency and ETS2 over-expression and suggest a distinct oncogenic potential for each factor. Overall, these results in primary murine fetal liver cells demonstrate that ETS2 and ERG1 promote the megakaryocyte lineage and facilitate immortalization of GATA1 knockdown cells, consistent with a potential oncogenic role in Down syndrome AMKL for these two ETS family genes located on chromosome 21.

Ets1 and Ets2 are nuclear phosphoproteins which bind to DNA in vitro and share two domains of strong identity. Deletion analyses of each of these conserved regions in Ets1 demonstrated that integrity of the carboxy-terminal domain, also conserved in the more distantly related elk and erg gene products, is essential for both nuclear targeting and DNA-binding activity in vitro.

Ets1 and Ets2 are nuclear phosphoproteins which bind to DNA in vitro and share two domains of strong identity. Deletion analyses of each of these conserved regions in Ets1 demonstrated that integrity of the carboxy-terminal domain, also conserved in the more distantly related elk and erg gene products, is essential for both nuclear targeting and DNA-binding activity in vitro.

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 Abstract 2561 We present the results of a study demonstrating that the genome profile of RUNX1 in MDS/AML is characterised by hitherto unreported partial deletions and absence of amplifications. This is in stark contrast to reports of chromosome 21 amplifications in ALL. We speculate that the absence of RUNX1 deletions results from them being well below a size detectable by commercial FISH probes. Extra chromosome 21 is the second most common acquired trisomy after (+) 8 in adult myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). It is rarely observed as sole abnormality but seen as part of complex karyotype in some 3–7% of the AML (Atlas of Genetics and Cytogenetics in Oncology and Haematology, http://atlasgeneticsoncology.org). Although the gene(s) in trisomy 21 associated with leukemia are unknown, the 21q22 region appears to be critical since it houses the RUNX1 gene. Multiple amplified copies of the RUNX1 carried by marker chromosomes, such as iAML21, are described in both acute lymphoblastic leukemia (ALL) and AML. A common 5.1 Mb amplification containing the RUNX1, miR-802 and genes mapping to the Down syndrome critical region identified in 91 children with iAML21, was shown to be the likely initiating event in this rare form of childhood B-cell ALL (Rand et al., Blood, 2011). In contrast, recent studies of AML in a Down syndrome and a constitutionally normal individual showed lack of RUNX1, ETS2 and ERG involvement (Canzonetta et al., BJH, 2012). Here we present 16 MDS/AML cases with imbalances of chromosome 21 identified by genomic array screening from a cohort of 83 cases. Whole genome screening (aCGH) was performed on presentation samples of MDS /AML and de novo AML cases using an oligonucleotide array platform (Agilent) at 60K, 244K, 400K and 1M density. G banding and FISH analysis were also successfully performed. Gain of an extra copy (trisomy) of chromosome 21 (+21) was found in 9 patients, all but one with complex karyotypes. In 2 AMLs high level amplifications were detected at 21q22, which involved the ETS2 and ERG but not the RUNX1 sequences. While several commercially available RUNX1 FISH probes showed gene multiple signals, custom FISH probes covering the relevant regions confirmed that the amplifications excluded the RUNX1 but affected both EST2 and ERG thus rendering the commercial probes unfit to assess CNA in this genome area. In another two cases with trisomy 12, cryptic loss of 43Kb and 98Kb resp. within the RUNX1 sequences was detected and confirmed by FISH. Furthermore, similar deletions within the 21q22.12 were also found in another 7 cases all of which had diploid set of chromosome 21 but had multiple changes at G banding level and high TGA score. These RUNX1 deletions were variable in size, ranging from 98Kb to 2.7Mb. Although our observations excluded clinical correlations it is note worthy that most of the patients with RUNX1 loss have not achieved complete cytogenetic remission. These findings suggest role for the RUNX1 loss as indicator of progressive disease and provide a novel insight into pathogenesis of MDS/AML. Disclosures: No relevant conflicts of interest to declare.

In the past years, the improvements in sequencing technology led to the development of “Next Generation Sequencing” (NGS) technologies. Several NGS approaches exist. Whole genome sequencing (WGS) and whole exome sequencing (WES) allow the identification of genomic alterations such as small insertions/deletions, point mutations and structural variants. Whole transcriptome sequencing (RNA-Seq) permits to quantify gene expression profiles and to detect alternative splicing and fusion transcripts. Recently, by using WES on atypical chronic myeloid leukemia (aCML) samples, our group identified recurrent mutations in SETBP1 gene; also, by using RNA-Seq on acute myeloid leukemia (AML), we identified a new fusion gene: ETS2-ERG. In aCML, SETBP1 mutations disrupt a degron binding site, leading to a decreased protein degradation. This leads to an increased amount of SETBP1 protein interacting with its natural ligand SET, which in turn acts inhibiting the protein phosphatase 2A (PP2A) oncosuppressor. Interestingly, the SETBP1 mutational cluster affected in aCML is highly conserved and the same mutations were also observed in the Schinzel-Giedion syndrome (SGS). However, the inhibition of the PP2A by SET, the only known interactor of SETBP1, does not explain the phenotype of SGS. To further characterize the role of SETBP1 protein, 293 Flp-In isogenic cellular models expressing the empty vector or the wild type (WT) or mutated (G870S) form of SETBP1 were established. In these models SETBP1 was fused with a V5 tag. Chromatin Immunoprecipitation sequencing experiments (Chip-Seq) performed against V5 confirmed the binding of SETBP1 to DNA, both for the WT and G870S forms. In addition, RNA-Seq experiments were performed. The comparison between Chip-Seq and RNA-Seq data has allowed us to identify 130 genes presenting both the binding of SETBP1 to their promoter region and transcriptional upregulation. Together these data suggest a role for SETBP1 as a transcriptional activator. Co-immunoprecipitation (Co-IP) experiments in transiently transfected HEK293T cells coupled with mass spectrometry (MS) analysis were performed to identify potential interactors of SETBP1. MS analysis led to the identification of the host cell factor 1 (HCF1), a component of the SET1/KMT2A COMPASS-like complex. Independent validation by western blot and fluorescence resonance energy transfer (FRET) confirmed the direct binding of HCF1 to SETBP1. Further independent experiments confirmed the Co-Ip of SET1/KMT2A and PHF8 with SETBP1. SET1/KMT2A is a core component of COMPASS-like complex and possesses H3K4 methyltransferase activity, whereas PHF8 possesses H4K20 demethylase activity. Both marks are associated with actively transcribed genes. Taken together, we have shown that SETBP1 protein is able to act as a transcriptional activator recruiting the HCF1/KMT2A/PHF8 complex. In a previous study, comparing cytogenetic analysis and RNA-Seq to detect chromosomal abnormalities on AML patient samples, a new fusion between the ETS2 and ERG genes was reported. The patient carrying this fusion was affected by acute promyelocytic leukemia (APL) and did not respond to therapy with retinoic acid. The role of the ETS2-ERG fusion is not known. To gain insight about the functional role of ETS2-ERG fusion in APL two cellular models were established. HL-60 and NB-4 cells were stable transfected with retroviral empty vector or with a vector carrying the fusion gene. This vector also carries the GFP as a positive selection marker. HL-60 cells carrying the ETS2-ERG fusion treated with retinoic acid showed a decrease in the expression at membrane level of the differentiation marker CD11b. This suggests that the ETS2-ERG fusion is able to impair the differentiation of APL cells upon retinoic acid treatment. Further experiments are ongoing to confirm the data in the NB4 cellular model.