Member Of Ets Family Research Articles

FLI1 is associated with regulation of DNA methylation and megakaryocytic differentiation in FPDMM caused by a RUNX1 transactivation domain mutation

Familial platelet disorder with associated myeloid malignancies (FPDMM) is an autosomal dominant disease caused by heterozygous germline mutations in RUNX1. It is characterized by thrombocytopenia, platelet dysfunction, and a predisposition to hematological malignancies. Although FPDMM is a precursor for diseases involving abnormal DNA methylation, the DNA methylation status in FPDMM remains unknown, largely due to a lack of animal models and challenges in obtaining patient-derived samples. Here, using genome editing techniques, we established two lines of human induced pluripotent stem cells (iPSCs) with different FPDMM-mimicking heterozygous RUNX1 mutations. These iPSCs showed defective differentiation of hematopoietic progenitor cells (HPCs) and megakaryocytes (Mks), consistent with FPDMM. The FPDMM-mimicking HPCs showed DNA methylation patterns distinct from those of wild-type HPCs, with hypermethylated regions showing the enrichment of ETS transcription factor (TF) motifs. We found that the expression of FLI1, an ETS family member, was significantly downregulated in FPDMM-mimicking HPCs with a RUNX1 transactivation domain (TAD) mutation. We demonstrated that FLI1 promoted binding-site-directed DNA demethylation, and that overexpression of FLI1 restored their megakaryocytic differentiation efficiency and hypermethylation status. These findings suggest that FLI1 plays a crucial role in regulating DNA methylation and correcting defective megakaryocytic differentiation in FPDMM-mimicking HPCs with a RUNX1 TAD mutation.

Abstract A015: Exploiting endogenous replication stress with a novel targeted therapy in Ewing sarcoma

Abstract Ewing sarcoma (EwS) is a rare and aggressive disease that typically affects the bone of children and young adults. Although there are moderate 5-year survival rates for primary disease, there is a high rate of recurrence and a lack of targeted therapies in this setting. Furthermore, pediatric exposure to chemotherapies leads to an increased risk of long-term complications, including secondary cancers, cardiomyopathy, and infertility. Therefore, there is a desperate need for targeted therapies which improve therapeutic outcomes and reduce exposure to chemotherapy in these patients. Like many pediatric cancers, EwS has a low mutational burden. Instead, it is driven by chromosomal translocation between a FET family member, EWSR1, and an ETS family member, usually FLI1. The EWSR1-FLI1 fusion protein acts as an aberrant and oncogenic transcription factor, disrupting cell cycle control, cell migration, and proliferation. ETS family members in these fusions retain their ability to bind to the SWI/SNF chromatin remodeling complex, further dysregulating DNA methylation and gene expression. Although targeting of the EWSR1 fusion has not yet been successful, these elevated levels of replication stress (RS) present a unique element of vulnerability within the cell. To this end, our lab has previously identified replication stress response (RSR) inhibitors, DDKi and WEE1i, which can be utilized to exploit the characteristic ability of EwS to maintain high levels of replication stress (RS) and push EwS cells into mitotic catastrophe in vitro. Here, we evaluate the efficacy of the replication stress response inhibitors (RSRi) TAK931 (DDKi) and MK1775 (WEE1i) with chemotherapy regimens commonly utilized in the relapsed/refractory setting in vivo. Using cell line derived xenografts in NSG mice, we show that DDKi and WEE1i with irinotecan limits tumor growth significantly more than existing therapy (temozolomide + irinotecan). Dosing schedules are optimized through dosing de-escalation and alternative-day dosing strategies. We further demonstrate the ability of our RSRi combination to effectively limit tumor growth over time in vivo even when irinotecan dosage is decreased. We go on to investigate the RSRi combination in the presence of ultra-low dose irinotecan. Finally, we utilize reduced representation bisulfite sequencing to examine the epigenetic effects of these treatment regimens. Overall, the exploitation of high endogenous replication stress in EwS by targeted RSRi presents a promising potential therapeutic option for this pediatric patient population. Citation Format: Emily Isenhart, Ajay Gupta, Bryan Gillard, Kristopher Attwood, Joyce Ohm. Exploiting endogenous replication stress with a novel targeted therapy in Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr A015.

Abstract 1099: Single-cell Transcriptomic And Epigenic Profiling Identifies Sox6 As A Key Regulator Of Arterial Endothelial Cell Phenotype In Systemic Sclerosis-associated Pulmonary Hypertension

Background: Pulmonary hypertension (PH) frequently arises as a complication of systemic sclerosis (SSc), and is a major cause of death among patients with this condition. Pulmonary vasculopathy in SSc is characterized by endothelial dysfunction with subsequent remodeling of the pulmonary vasculature, especially arterioles and small arteries. Characterization of transcriptional regulators driving the pathological changes in arterial endothelial cells (ECs) is critical to understanding pathogenic mechanisms underlying SSc-associated vasculopathy. Methods and Results: We performed single-cell multiome sequencing to simultaneously profile the transcriptome and epigenome of explanted lung tissue from SSc-PH patients (n = 9) and healthy donors (n = 6). We identified co-regulated genes and candidate enhancer regions associated with transcription factors (TFs) across different EC cell states using SCENIC+. Gene regulatory network analysis revealed SOX6 as a key transcription factor for the arterial EC phenotype associated with SSc-PH. 71 genes and 153 regions were predicted to be regulated by SOX6. Notably, SOX6 was the only SOX family member significantly upregulated in SSc-PH arterial ECs (fold change = 40.0, adjusted p-value = 6.1*10 -21 ). Putative bound TF motif instances accounting for chromatin accessibility in the candidate enhancer regions were identified using the base-resolution deep learning model, ChromBPNet. Consistent with SCENIC+ predictions, cell-state specific ChromBPNet models revealed higher putative TF occupancy at SOX motif instances across the genome. In addition, we identified SOX/ETS composite motif instances showing higher putative occupancy in SSc-PH arterial ECs, which may support cooperative binding of SOX proteins with ETS family members. Conclusions: These data provide novel insight into transcriptional regulatory programs of arterial ECs in SSc-PH and highlight SOX6 as a key contributor.

SPDEF drives pancreatic adenocarcinoma progression via transcriptional upregulation of S100A16 and activation of the PI3K/AKT signaling pathway.

Pancreatic adenocarcinoma (PAAD) is a notably aggressive malignancy with limited treatment options and an unfavorable prognosis for patients. We aimed to investigate molecular mechanisms by which Sam's pointed domain-containing ETS transcription factor (SPDEF) exerts effects on PAAD progression. We analyzed differentially expressed genes (DEGs) and their integration with ETS family members using the The Cancer Genome Atlas (TCGA) database, hence identifying SPDEF as a core gene in PAAD. Kaplan-Meier survival analysis confirmed SPDEF's prognostic potential. In vitro experiments validated the association with cell proliferation and apoptosis, affecting pancreatic cancer cell dynamics. We detected increased SPDEF expression in PAAD tumor samples. Our in vitro studies revealed that SPDEF regulates mRNA and protein expression levels, and significantly affects cell proliferation. Moreover, SPDEF was associated with reduced apoptosis and enhanced cell migration and invasion. In-depth analysis of SPDEF-targeted genes revealed four crucial genes for advanced prognostic model, among which S100A16 was significantly correlated with SPDEF. Mechanistic analysis showed that SPDEF enhances the transcription of S100A16, which in turn enhances PAAD cell migration, proliferation, and invasion by activating the PI3K/AKT signaling pathway. Our study revealed the critical role of SPDEF in promoting PAAD by upregulating S100A16 transcription and stimulating the PI3K/AKT signaling pathway. This knowledge deepened our understanding of pancreatic cancer's molecular progression and unveiled potential therapeutic strategies targeting SPDEF-driven pathways.

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.

Cohesin Haploinsufficiency Promotes Reduced Latency in Inv(16) Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a cancer of the bone marrow that affects 20,000 adults in the United States alone, with global rates increasing over the last 30 years (Heimbruch et al. 2021, Dong et al. 2020). Approximately 15% of adult AML patients harbor the chromosomal aberrations t(8;21) or inv(16), which affect the core binding complex members RUNX1 and CBFB, respectively. Together, RUNX1 and CBFB function as a transcription factor that regulates key lineage genes and proliferation in hematopoietic cells (Pulikkan and Castilla 2018). t(8;21) and inv(16) result in the expression of the fusion proteins AML1-ETO and CBFB-SMMHC, both of which have dominant negative effects on native RUNX1 as well as novel transcriptional roles (Surapally et al. 2021). Although both t(8;21) and inv(16) result in AMLs with similar characteristics, they differ in the number and type of co-occurring mutations (Opatz et al. 2020). More mutations per clone are observed in t(8;21) vs. inv(16) AMLs. Additionally, several studies have highlighted a difference between the rates of mutation of cohesin genes and t(8;21) vs. inv(16) (Faber et al. 2016, Opatz et al. 2020, Duployez et al. 2016, Jahn et al. 2020, Qin et al. 2022). Cohesin is comprised of the subunits SMC3, SMC1A, RAD21, and STAG2. The complex has a prominent role in hematopoietic cells in chromatin organization and DNA looping events that regulate gene expression and self-renewal (Heimbruch et al. 2021). Cohesin mutations are found in approximately 10% of AML cases (Ley et al 2013). Interestingly, cohesin mutations are found in 18-27% of t(8;21) AMLs, but in 0-4% of inv(16) AMLs (Faber et al. 2016, Opatz et al. 2020, Duployez et al. 2016, Jahn et al. 2020, Qin et al. 2022). The low rate of co-mutation between inv(16) and cohesin genes suggests potential synthetic lethality. However, it is also possible that cohesin mutations occur at a higher rate than reported due to biases in the age, ethnicity, or treatment status of the patients included in genome/exome sequencing studies. To test if cohesin mutations and inv(16) are synthetic lethal, we used an inv(16) knock-in mouse model ( Mx1-Cre;inv(16)) combined with a cohesin-deficient mouse ( Mx1-Cre; Smc3 fl/+) (Kuo et al. 2006, Viny et al. 2015). We followed inv(16);Smc3wt and inv(16);Smc3 -/+ animals post-Cre activation and monitored mice for leukemic development. We found that inv(16);Smc3 -/+ mice developed AML with reduced latency compared to inv(16);Smc3wt mice, suggesting cooperativity rather than synthetic lethality. To determine the mechanism by which Smc3 haploinsufficiency accelerates inv(16) leukemias, we performed ATAC sequencing and single cell RNA sequencing on hematopoietic stem and progenitor cells (HSPCs). ATAC-seq revealed a large increase in open chromatin in promoter regions in inv(16);Smc3 -/+ vs. inv(16);Smc3wt HSPCs. These open regions were enriched for binding motifs of the Ets family member Fli1. Increased expression of Fli1 was observed by western blotting in inv(16);Smc3 -/+ HSPCs. Interestingly, CBFB-SMMHC binding sites commonly contain Ets motifs (Mandoli 2014), suggesting that the increased Fli1 expression and binding site accessibility in inv(16);Smc3 -/+ may drive the observed decrease in latency. scRNA-seq revealed cluster-specific differences between inv(16);Smc3wt and inv(16);Smc3 -/+ cells. In several clusters, inv(16);Smc3 -/+ cells showed a loss of expression of genes involved in apoptosis and differentiation. Of particular interest is Gata2, as loss of Gata2 generates a more aggressive phenotype in AML (Saida et al. 2020). Therefore, Smc3 haploinsufficiency may result in Gata2 deregulation, thereby impacting disease latency. Currently, we have two potential explanations for the observed phenotype: Deregulated binding of Fli1 and its downstream transcriptional pathways and decreased expression of Gata2 in sub-populations of inv(16);Smc3 -/+ cells. Which of these mechanisms mediates the reduced latency in inv(16);Smc3 -/+ animals is an area of active investigation. Collectively, our studies show that a lack of observed co-mutation does not necessarily indicate synthetic lethality. We propose that cohesin mutations may be more common in inv(16) leukemias than has been reported and suggest that a careful consideration of age, ethnicity, and treatment status may be warranted when evaluating rates of co-mutation.

Adamantinoma-like Ewing Sarcoma (ALES) May Harbor FUS Rearrangements : A Potential Diagnostic Pitfall.

Adamantinoma-like Ewing sarcoma (ALES) is a rare malignancy currently considered a variant of Ewing sarcoma with most known cases harboring EWSR1 rearrangements. Herein we present a series of 6 cases of EWSR1 -negative ALES. The tumors arose in the sinonasal tract (n=3), major salivary glands (submandibular gland=1; parotid=1), and anterior mediastinum (n=1) in patients ranging from 25 to 79 years of age. Most tumors were basaloid in appearance, growing in large nests separated by interlobular fibrosis without overt squamous pearls. However, 1 case closely resembled a well-differentiated neuroendocrine tumor with uniformly round nuclei, eosinophilic cytoplasm, and trabecular architecture. All cases were diffusely positive for pan-cytokeratin, p40 or p63, and CD99. A subset of cases showed diffuse reactivity for synaptophysin, including 1 sinonasal tumor which also demonstrated sustentacular S100 protein expression. Molecular testing showed FUS rearrangements in all cases. Gene partners included known ETS family members FEV (n=2) and FLI1 (n=1). Our results expand the molecular diagnostic considerations for ALES to include FUS rearrangements. We also show that ALES may harbor FUS :: FLI1 fusion, which has not been previously reported in the Ewing family of tumors. Furthermore, ALES may show unusual histologic and immunophenotypic features that can overlap with olfactory carcinoma including S100-positive sustentacular cells. ALES should be considered in the diagnostic differential of small round cell tumors and tumors with neuroendocrine differentiation with immunohistochemical workup to include p40 and CD99/NKX2.2.

Abstract 1467: A novel screening model to identify EWS::FLI1 regulators and their consequences on Ewing sarcoma plasticity

Abstract Ewing sarcoma (ES) is a rare, highly aggressive cancer of the bone and soft tissues that mainly affects children and young adults. The most common driver of ES is a chromosomal translocation between the EWSR1 gene and an ETS family member, most frequently FLI1. This leads to the expression of an aberrant transcription factor called EWS::FLI1 (EF1) that acts as an epigenetic reprogrammer and transcriptional modulator. Previous studies on ES cell lines showed that EF1 levels are dynamic where cells with high EF1 levels are more proliferative, while cells with low EF1 levels have a strong propensity to migrate and metastasize. However, there is no direct evidence of EF1 fluctuations on a functional protein level. Moreover, the upstream modulators or pathways regulating EF1 thresholds and their corresponding consequences on ES tumorigenesis are poorly understood. Thus, we aimed to develop an ES screening model to identify EF1 regulators by performing a high-throughput drug screen and a genome-wide CRISPR/Cas9 knockout screen. This model was established by endogenously tagging EF1 of ES cell lines (A673, TC71) with a fluorescent tag (mNeonGreen) together with a degradation tag (dTAG) using CRISPR/Cas9 knock-in. The fluorescent tag serves for the readout of altered EF1 expression on multiple levels (transcriptional, post-transcriptional, translational, post-translational, and epigenetic). At the same time, dTAG allows mimicking rapid and acute EF1 perturbation-specific cell states upon treatment with a VHL-recruiting ligand (dTAGv-1). The successful knock-in clones with edited EF1 were extensively characterized compared to the wildtype EF1 cells with respect to cellular morphology, subcellular EF1 localization, stability, and target gene expression to rule out a loss of EF1 function due to the addition of the tags. The results from the high throughput drug screen (2.5K custom compound library) resulted in a candidate list of EF1 up- and down-regulators involving genes of various cancer signaling pathways (PI3K/AKT/mTOR, IGF-1, ALK pathway), cell cycle regulators (CDKs, AURKA, PLK1) and epigenetic regulators (HDACs, BETs). In the next steps, the drug targets will be compared to the gene hits from the CRISPR screen in an unbiased manner to identify novel EF1-regulatory pathways. Moreover, the functional consequences of treatment with EF1-modulatory compounds on ES tumorigenesis will be studied. Together, our study will promote a better understanding of factors affecting EF1 thresholds and ES plasticity and help identify potential therapeutic targets for ES. Citation Format: Veveeyan Suresh, Valerie Fock, Beat Schäfer, Heinrich Kovar. A novel screening model to identify EWS::FLI1 regulators and their consequences on Ewing sarcoma plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1467.

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.

FGF19-mediated ELF4 overexpression promotes colorectal cancer metastasis through transactivating FGFR4 and SRC.

Background: Metastasis accounts for the high lethality of colorectal cancer (CRC) patients. Unfortunately, the molecular mechanism manipulating metastasis in CRC is still elusive. Here, we investigated the function of E74-like factor 4 (ELF4), an ETS family member, in facilitating CRC progression. Methods: The expression of ELF4 in human CRC samples and CRC cell lines was determined by quantitative real-time PCR, immunohistochemistry and immunoblotting. The migratory and invasive phenotypes of CRC cells were evaluated by in vitro transwell assays and in vivo metastatic models. The RNA sequencing was used to explore the downstream targets of ELF4. The luciferase reporter assays and chromatin immunoprecipitation assays were used to ascertain the transcriptional regulation related to ELF4. Results: We found elevated ELF4 was positively correlated with distant metastasis, advanced AJCC stages, and dismal outcomes in CRC patients. ELF4 expression was also an independent predictor of poor prognosis. Overexpression of ELF4 boosted CRC metastasis via transactivating its downstream target genes, fibroblast growth factor receptor 4 (FGFR4) and SRC proto-oncogene, non-receptor tyrosine kinase, SRC. Fibroblast growth factor 19 (FGF19) upregulated ELF4 expression through the ERK1/2/SP1 axis. Clinically, ELF4 expression had a positive correlation with FGF19, FGFR4 and SRC, and CRC patients who positively coexpressed FGF19/ELF4, ELF4/FGFR4, or ELF4/SRC exhibited the worst clinical outcomes. Furthermore, the combination of the FGFR4 inhibitor BLU-554 and the SRC inhibitor KX2-391 dramatically suppressed ELF4-mediated CRC metastasis. Conclusions: We demonstrated the essentiality of ELF4 in the metastatic process of CRC, and targeting the ELF4-relevant positive feedback circuit might represent a novel therapeutic strategy.

Generation of a doxycycline-inducible ETV2 expression cell line using PiggyBac transposase system

E26 transformation-specific variant 2 (ETV2), as a member of Ets family of transcription factors, plays an important role in embryonic vasculogenesis, angiogenesis and hematopoiesis. Here, based on H9 cell line and PiggyBac technology, we generated a doxycycline-inducible ETV2 (Dox-ETV2-H9) embryonic stem cell (ESC) line. The Dox-ETV2-H9 cells maintain normal morphology, karyotype as well as high expression of pluripotent markers. In comparison with previous studies that employing costly ETV2 modRNA to improve differentiation from ESCs into endothelial cells (ECs), the tet-on system in Dox-ETV2-H9 cells could precisely manipulate ETV2 expression, which enables ECs differentiation process more efficient and controllable upon addition of doxycycline.

The CIC-ERF co-deletion underlies fusion-independent activation of ETS family member, ETV1, to drive prostate cancer progression.

Human prostate cancer can result from chromosomal rearrangements that lead to aberrant ETS gene expression. The mechanisms that lead to fusion-independent ETS factor upregulation and prostate oncogenesis remain relatively unknown. Here, we show that two neighboring transcription factors, Capicua (CIC) and ETS2 repressor factor (ERF), which are co-deleted in human prostate tumors can drive prostate oncogenesis. Concurrent CIC and ERF loss commonly occur through focal genomic deletions at chromosome 19q13.2. Mechanistically, CIC and ERF co-bind the proximal regulatory element and mutually repress the ETS transcription factor, ETV1. Targeting ETV1 in CIC and ERF-deficient prostate cancer limits tumor growth. Thus, we have uncovered a fusion-independent mode of ETS transcriptional activation defined by concurrent loss of CIC and ERF.

Genome-wide interrogation of structural variation reveals novel African-specific prostate cancer oncogenic drivers

BackgroundAfrican ancestry is a significant risk factor for advanced prostate cancer (PCa). Mortality rates in sub-Saharan Africa are 2.5-fold greater than global averages. However, the region has largely been excluded from the benefits of whole genome interrogation studies. Additionally, while structural variation (SV) is highly prevalent, PCa genomic studies are still biased towards small variant interrogation.MethodsUsing whole genome sequencing and best practice workflows, we performed a comprehensive analysis of SVs for 180 (predominantly Gleason score ≥ 8) prostate tumours derived from 115 African, 61 European and four ancestrally admixed patients. We investigated the landscape and relationship of somatic SVs in driving ethnic disparity (African versus European), with a focus on African men from southern Africa.ResultsDuplication events showed the greatest ethnic disparity, with a 1.6- (relative frequency) to 2.5-fold (count) increase in African-derived tumours. Furthermore, we found duplication events to be associated with CDK12 inactivation and MYC copy number gain, and deletion events associated with SPOP mutation. Overall, African-derived tumours were 2-fold more likely to present with a hyper-SV subtype. In addition to hyper-duplication and deletion subtypes, we describe a new hyper-translocation subtype. While we confirm a lower TMPRSS2-ERG fusion-positive rate in tumours from African cases (10% versus 33%), novel African-specific PCa ETS family member and TMPRSS2 fusion partners were identified, including LINC01525, FBXO7, GTF3C2, NTNG1 and YPEL5. Notably, we found 74 somatic SV hotspots impacting 18 new candidate driver genes, with CADM2, LSAMP, PTPRD, PDE4D and PACRG having therapeutic implications for African patients.ConclusionsIn this first African-inclusive SV study for high-risk PCa, we demonstrate the power of SV interrogation for the identification of novel subtypes, oncogenic drivers and therapeutic targets. Identifying a novel spectrum of SVs in tumours derived from African patients provides a mechanism that may contribute, at least in part, to the observed ethnic disparity in advanced PCa presentation in men of African ancestry.

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.

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.

ERG Expression is Helpful in Differentiating T-Lymphoblastic Lymphoma from Thymoma.

ETS-related gene (ERG) is the member of ETS-family of transcription factors and is commonly expressed in Ewing sarcoma. Recently, we found that ERG can also be expressed in lymphoblastic lymphoma. The aim of this article is to explore the expression patterns of ERG in T-lymphoblastic lymphoma, and to evaluate its diagnostic value for differentiating T-lymphoblastic lymphoma and nonneoplastic T-precursor cells in thymoma via immunohistochemistry. In this study, we explored the expression pattern of ERG in T-lymphoblastic lymphoma and thymoma specimens via immunohistochemistry. Sixteen T-lymphoblastic lymphoma and 18 thymoma specimens were evaluated for the expression of ERG. Our findings showed that ERG was expressed in 10 of the 16 (63%) T-lymphoblastic lymphoma specimens, and in only 1 of the 18 (6%) thymoma specimens. The positive and negative predictive value of ERG in T-lymphoblastic lymphoma was 91% and 74%, respectively. ERG is a helpful marker for the diagnosis of T-lymphoblastic lymphoma and is a promising new method to differentiate T-lymphoblastic lymphoma and the nonneoplastic T-precursor cells in thymoma.

ETS factors in prostate cancer.

The ETS family of proteins consists of 28 transcription factors, many of which play critical roles in both normal tissue development and homeostasis and have been implicated in development and progression of a variety of cancers. In prostate cancer, gene fusion and overexpression of ETS factors ERG, FLI1, ETV1, ETV4 and ETV5 have been found in half of prostate cancer patients in Caucasian men and define the largest genetic subtype of prostate cancer. This review summarizes the data on the discovery, modeling, molecular taxonomy, lineage plasticity and therapeutic targeting of ETS family members in prostate cancer.

Past, Current, and Future Strategies to Target ERG Fusion-Positive Prostate Cancer.

Simple SummaryIn addition to its role in development and in the vascular and hematopoietic systems, ERG plays a central role in prostate cancer. Approximately 40–50% of prostate cancer cases are characterized by ERG gene fusions, which lead to ERG overexpression. Importantly, inhibition of ERG activity in prostate cancer cells decreases their viability. Therefore, inhibiting ERG might represent an important step to improve treatment efficacy for patients with ERG-positive prostate tumors. Here, we summarize the attempts made over the past years to repress ERG activity, the current use of ERG fusion detection and the strategies that might be utilized in the future to treat ERG fusion-positive tumors.The ETS family member ERG is a transcription factor with physiological roles during development and in the vascular and hematopoietic systems. ERG oncogenic activity characterizes several malignancies, including Ewing’s sarcoma, leukemia and prostate cancer (PCa). In PCa, ERG rearrangements with androgen-regulated genes—mostly TMPRSS2—characterize a large subset of patients across disease progression and result in androgen receptor (AR)-mediated overexpression of ERG in the prostate cells. Importantly, PCa cells overexpressing ERG are dependent on ERG activity for survival, further highlighting its therapeutic potential. Here, we review the current understanding of the role of ERG and its partners in PCa. We discuss the strategies developed in recent years to inhibit ERG activity, the current therapeutic utility of ERG fusion detection in PCa patients, and the possible future approaches to target ERG fusion-positive tumors.

Implementation of a prostate cancer-specific targeted sequencing panel for credentialing of patient-derived cell lines and genomic characterization of patient samples.

Primary and metastatic prostate cancers have low mutation rates and recurrent alterations in a small set of genes, enabling targeted sequencing of prostate cancer-associated genes as an efficient approach to characterizing patient samples (compared to whole-exome and whole-genome sequencing). For example, targeted sequencing provides a flexible, rapid, and cost-effective method for genomic assessment of patient-derived cell lines to evaluate fidelity to initial patient tumor samples. We developed a prostate cancer-specific targeted next-generation sequencing (NGS) panel to detect alterations in 62 prostate cancer-associated genes as well as recurring gene fusions with ETS family members, representing the majority of common alterations in prostate cancer. We tested this panel on primary prostate cancer tissues and blood biopsies from patients with metastatic prostate cancer. We generated patient-derived cell lines from primary prostate cancers using conditional reprogramming methods and applied targeted sequencing to evaluate the fidelity of these cell lines to the original patient tumors. The prostate cancer-specific panel identified biologically and clinically relevant alterations, including point mutations in driver oncogenes and ETS family fusion genes, in tumor tissues from 29 radical prostatectomy samples. The targeted panel also identified genomic alterations in cell-free DNA and circulating tumor cells (CTCs) from patients with metastatic prostate cancer, and in standard prostate cancer cell lines. We used the targeted panel to sequence our set of patient-derived cell lines; however, no prostate cancer-specific mutations were identified in the tumor-derived cell lines, suggesting preferential outgrowth of normal prostate epithelial cells. We evaluated a prostate cancer-specific targeted NGS panel to detect common and clinically relevant alterations (including ETS family gene fusions) in prostate cancer. The panel detected driver mutations in a diverse set of clinical samples of prostate cancer, including fresh-frozen tumors, cell-free DNA, CTCs, and cell lines. Targeted sequencing of patient-derived cell lines highlights the challenge of deriving cell lines from primary prostate cancers and the importance of genomic characterization to credential candidate cell lines. Our study supports that a prostate cancer-specific targeted sequencing panel provides an efficient, clinically feasible approach to identify genetic alterations across a spectrum of prostate cancer samples and cell lines.

EHF is essential for epidermal and colonic epithelial homeostasis, and suppresses Apc-initiated colonic tumorigenesis.

Ets homologous factor (EHF) is a member of the epithelial-specific Ets (ESE) family of transcription factors. To investigate its role in development and epithelial homeostasis, we generated a series of novel mouse strains in which the Ets DNA-binding domain of Ehf was deleted in all tissues (Ehf-/-) or specifically in the gut epithelium. Ehf-/- mice were born at the expected Mendelian ratio, but showed reduced body weight gain, and developed a series of pathologies requiring most Ehf-/- mice to reach an ethical endpoint before reaching 1 year of age. These included papillomas in the facial skin, abscesses in the preputial glands (males) or vulvae (females), and corneal ulcers. Ehf-/-mice also displayed increased susceptibility to experimentally induced colitis, which was confirmed in intestinal-specific Ehf knockout mice. Gut-specific Ehf deletion also impaired goblet cell differentiation, induced extensive transcriptional reprogramming in the colonic epithelium and enhanced Apc-initiated adenoma development. The Ets DNA-binding domain of EHF is therefore essential for postnatal homeostasis of the epidermis and colonic epithelium, and its loss promotes colonic tumour development.