Wild-type ERG Research Articles

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

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

ERG Is a New Predisposition Gene for Bone Marrow Failure and Hematological Malignancy

There remain gaps in our knowledge of hereditary and sporadic causes of hematological malignancy (HM) and bone marrow failure (BMF) that prevent optimal diagnosis, disease surveillance and treatment. Here we report the discovery of ERG as a novel predisposition gene for BMF and HM. ERG is a known oncogene, typically via gene-fusions, leading to dysregulated ERG overexpression in blood and solid cancers. We identified a germline ERG ETS domain variant p.Y373C segregating with thrombocytopenia in a mother, who progressed to AML (27 yr) and then therapy-related MDS (35 yr), and in her 2 sons. All three showed copy neutral loss of heterozygosity of all or part of chromosome 21q, including the ERG locus, with the oldest son showing at least 2 somatic genetic rescue (SGR) events. The possibility of causal RUNX1 variants were ruled out, with the smallest somatic cnLOH event beginning within the RUNX1 gene, but not encompassing the RUNT domain where the majority of pathogenic missense variants are located. ERG, a highly constrained gene (LOEUF <0.33), is critical for definitive hematopoiesis, adult hematopoietic stem cell (HSC) function and platelet maintenance. An identical corresponding heterozygous germline variant (p.Y343C) in ERG’s closest gene by homology, FLI1, causes platelet-type bleeding disorder-21 (BDPLT21, OMIM #617443). Through global collaborations, we have identified 15 heterozygous variants in the ERG gene, 13 of which are missense and 2 truncating variants, in 17 individuals with cytopenia and/or HM (mainly myeloid) or lymphedema (Table). Onset of hematological symptoms ranged from birth to 38 years for truncating and constrained ETS domain variants. Of these 15 variants, 12 have been confirmed germline including 2 de novo. Only 4 meiotic transmissions are observed. None of the missense variants in the highly conserved ETS domain of ERG which mediates DNA binding, protein-protein interactions and nuclear localization, are present in gnomAD. We have functionally characterized 19 ERG variants, 12 potentially pathogenic, 1 known mouse pathogenic variant and 3 population controls demonstrating that most ETS domain missense variants display loss-of-function (LOF) characteristics disrupting transcriptional transactivation (Figure), DNA-binding and/or nuclear localization in vitro. Robust preliminary data from ex vivo models of ERG overexpression in mouse fetal liver cells in tissue culture (cytokine-independence), a mouse transplant assay and previous germline mutant Erg mouse models are concordant with ETS domain missense variants being LOF compared to wildtype ERG and benign controls. Together, these data provide clinical, in vitro and ex vivo functional studies implicating LOF variants in hematological disease predisposition. LOF ERG mutations also occur in sporadic cases of HM. Recently, as part of a Genomics England Research Consortium population study, 4 truncating ERG variants were described in 7 individuals across 4 families with 3 meiotic transmissions and a de novo case with primary lymphedema (1) and we add 2 novel missense variants here. One patient showed SGR across the ERG locus in blood. Blood phenotypes were not described. Our results demonstrate that germline ERG variants predispose to diverse cytopenia, BMF and HM in both children and adults. In our family mentioned above, the mother received an unrelated alloHSCT due to t-MDS while her 2 sons with cytopenias continue to be monitored. The natural history of this new syndrome will require careful identification of germline lesions with additional longitudinal studies in more patients and families needed. This ERG syndrome parallels GATA2 deficiency syndrome (HM and lymphedema) and RUNX1 Familial Platelet disorder-myeloid malignancy (thrombocytopenia and HM). Like the well-known disease genes GATA2 and RUNX1, ERG is also a member of the transcription factor heptad involved in HSC maintenance and differentiation. ERG adds to a growing list of genes whose unregulated expression contributes to HM and other cancers. Identification of causal germline ERG variants like those outlined in this study, has direct clinical implications for patient and family management including diagnosis, counselling, surveillance and treatment strategies such as selection of bone marrow transplant donors and potential for targeted therapies including gene and cell therapy. Reference 1. Greene D et al. Nat.Med. 29:679-688 2023

Prognostic Utility of Biopsy-Based PTEN and ERG Status on Biochemical Progression and Overall Survival after SBRT for Localized Prostate Cancer

<h3>Purpose/Objective(s)</h3> PTEN deletions and ERG rearrangements are two of the most common genetic abnormalities associated with prostate cancer. Prior studies revealed that changes in wild-type PTEN and ERG portend worse clinical outcomes. While early studies were performed on whole-gland samples after radical prostatectomy, subsequent biopsy-based analyses have led to their inclusion in several genomic assays and prostate cancer outcome scores. However, evaluations of therapeutic response to radiotherapy are quite heterogenous. Our objective is to evaluate the impact of biopsy-determined <i>PTEN</i> losses and <i>TMPRSS2-ERG</i> fusion on biochemical disease-free survival (bDFS) in patients who receive SBRT for localized prostate cancer. <h3>Materials/Methods</h3> Patients included in this analysis underwent SBRT for localized prostate cancer and are followed on an institutional prospective quality of life and survival study. For each patient, the single biopsy core with the highest volume of cancer was evaluated for PTEN and ERG abnormalities. Differences in baseline patient and disease characteristics between groups were analyzed using ANOVA for age and χ<b>²</b> for categorical groupings. bDFS and OS were calculated using the Kaplan Meier (KM) method with Log-Rank test comparison between groups. Predictors of bDFS and OS were evaluated using Cox proportional hazard method. For all analyses, <i>p</i> <0.05 was considered statistically significant. <h3>Results</h3> Ninety-three patients were included in the analysis with a median follow up of 72 months (IQR 52 - 85 months). The 5-year bDFS for all patients was 85.2% with a median bDFS of 98 months. The 5-year OS for all patients was 91.8% with a median OS of 123 months. Differences between ERG and PTEN status groups by age, race, T-stage, NCCN risk group, PSA group, PSA density group, and Charlson Comorbidity Index were not statistically significant. Differences in grade group were statistically significant (<i>p</i> = 0.0329). The estimated 5-year bDFS was 100%, 76.6%, 72.9%, and 63.8% for the ERG+/PTEN, ERG-/PTEN+, ERG+/PTEN-, and ERG-/PTEN- groups respectively (<i>p</i> =0.0107). Multivariate analysis (MVA) confirmed an increased likelihood of 5-year biochemical progression with higher NCCN risk group (<i>p</i> <0.0001) and increasing ERG/PTEN mutational burden (<i>p</i><0.0001). <h3>Conclusion</h3> <i>ERG Fusion with PTEN</i> loss is associated with an increased risk of biochemical progression. However, <i>ERG</i> status alone is not predictive of bPFS. Biopsy-based PTEN and ERG assays demonstrate utility in predicting 5-year oncologic outcomes in patients who receive prostate SBRT.

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

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

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&amp;lt;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&amp;lt;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.

ERG transcription factors have a splicing regulatory function involving RBFOX2 that is altered in the EWS-FLI1 oncogenic fusion

ERG family proteins (ERG, FLI1 and FEV) are a subfamily of ETS transcription factors with key roles in physiology and development. In Ewing sarcoma, the oncogenic fusion protein EWS-FLI1 regulates both transcription and alternative splicing of pre-messenger RNAs. However, whether wild-type ERG family proteins might regulate splicing is unknown. Here, we show that wild-type ERG proteins associate with spliceosomal components, are found on nascent RNAs, and induce alternative splicing when recruited onto a reporter minigene. Transcriptomic analysis revealed that ERG and FLI1 regulate large numbers of alternative spliced exons (ASEs) enriched with RBFOX2 motifs and co-regulated by this splicing factor. ERG and FLI1 are associated with RBFOX2 via their conserved carboxy-terminal domain, which is present in EWS-FLI1. Accordingly, EWS-FLI1 is also associated with RBFOX2 and regulates ASEs enriched in RBFOX2 motifs. However, in contrast to wild-type ERG and FLI1, EWS-FLI1 often antagonizes RBFOX2 effects on exon inclusion. In particular, EWS-FLI1 reduces RBFOX2 binding to the ADD3 pre-mRNA, thus increasing its long isoform, which represses the mesenchymal phenotype of Ewing sarcoma cells. Our findings reveal a RBFOX2-mediated splicing regulatory function of wild-type ERG family proteins, that is altered in EWS-FLI1 and contributes to the Ewing sarcoma cell phenotype.

DNA Damage Promotes TMPRSS2-ERG Oncoprotein Destruction and Prostate Cancer Suppression via Signaling Converged by GSK3β and WEE1

TMPRSS2-ERG gene fusion occurs in approximately 50% of cases of prostate cancer (PCa), and the fusion product is a key driver of prostate oncogenesis. However, how to leverage cellular signaling to ablate TMPRSS2-ERG oncoprotein for PCa treatment remains elusive. Here, we demonstrate that DNA damage induces proteasomal degradation of wild-type ERG and TMPRSS2-ERG oncoprotein through ERG threonine-187 and tyrosine-190 phosphorylation mediated by GSK3β and WEE1, respectively. The dual phosphorylation triggers ERG recognition and degradation by the E3 ubiquitin ligase FBW7 in a manner independent of a canonical degron. DNA damage-induced TMPRSS2-ERG degradation was abolished by cancer-associated PTEN deletion or GSK3β inactivation. Blockade of DNA damage-induced TMPRSS2-ERG oncoprotein degradation causes chemotherapy-resistant growth of fusion-positive PCa cells in culture and in mice. Our findings uncover a previously unrecognized TMPRSS2-ERG protein destruction mechanism and demonstrate that intact PTEN and GSK3β signaling are essential for effective targeting of ERG protein by genotoxic therapeutics in fusion-positive PCa.

SPOP and FOXA1 mutations are associated with PSA recurrence in ERG wt tumors, and SPOP downregulation with ERG-rearranged prostate cancer.

ERG fusion-related prostate cancer (PrCa) is the most prevalent oncogenic driver subclass. SPOP, FOXA1, and IDH1 mutations are other three main oncogenic driver subclasses in non-ETS-fusion PrCa. ERG protein levels seem to be increased in SPOP-mutated cases, and different studies reported that SPOP mutations and ERG fusions are mutually exclusive. The aim of this study has been to analyze the alterations in non-ETS-oncogenic drivers in PrCa. SPOP, FOXA1, and IDH mutations were investigated by polymerase chain reaction (PCR) and Sanger direct sequencing. ERG, SPOP, and TMPRSS2-ERG messenger RNA expression was assessed by quantitative real-time PCR from complementary DNA, and the presence of the fusion was also analyzed by nonquantitative PCR. The clinical pathological features were retrieved from the charts of the 111 patients included in the study (MARBiobanc, Barcelona, Spain). Loss of SPOP expression (25.2%) was associated with ERG overexpression (P = 0.0036). SPOP mutations were found in 5.4% cases, all with wild-type (wt) ERG (P = 0.007). FOXA1 mutations were found in 8.2% cases, most of them ERG wt (P = 0.06). No IDH1 mutations were found. SPOP or FOXA1 mutations were found in 1.7% of ERG-rearranged, and 34.2% of non-ERG-rearranged cases (P < 0.0001). SPOP or FOXA1 alterations (mutations or expression loss) were significantly more common in GG5, while isolated ERG overexpression was more common in GG1 tumors (P = 0.042). SPOP-or FOXA1-mutated cases were associated with a shorter time to prostate-specific antigen (PSA) recurrence in the univariate (P = 0.0009), and with the PSA recurrence risk in the multivariate (P = 0.023) analysis. In conclusion, SPOP and FOXA1 mutations may have prognostic value in ERG wt tumors. Interestingly, in absence of SPOP mutations, downregulation of this gene is a feature of many ERG-rearranged prostate tumors.

Association of ERG/PTEN status with biochemical recurrence after radical prostatectomy for clinically localized prostate cancer.

We have previously demonstrated a significant correlative relationship between PTEN deletion and ERG rearrangement, both in the development of clinically localized prostate cancers and metastases. Herein, we evaluate the cooperative role of ERG and PTEN in oncological outcomes after radical prostatectomy for clinically localized prostate cancer. We evaluated ERG and PTEN status using three previously described cohorts. The first cohort included 235 clinically localized prostate cancer cases represented on tissue microarrays (TMA), evaluated using previously validated FISH assays for ERG and PTEN. The second cohort included 167 cases of clinically localized prostate cancer on TMAs evaluated for PTEN by FISH, and for PTEN and ERG by dual IHC. The third cohort comprised 59 clinically localized prostate cancer cases assessed by array comparative genomic hybridization (aCGH). Kaplan-Meir plots and long rank tests were used to assess the association of ERG and PTEN status with biochemical recurrence after radical prostatectomy for clinically localized prostate cancer. Of the 317 cases eligible for analyses with evaluable ERG and PTEN status, 88 (27.8%) patients developed biochemical recurrence over a median follow-up of 5.7years. Overall, 45% (142/317) of cases demonstrated ERG rearrangement and 20% (62/317) of cases demonstrated PTEN loss. Hemizygous and homozygous deletion of PTEN was seen in 10% (18/175) and 3% (5/175) of ERG-negative cases, respectively. In contrast, hemizygous and homozygous deletion of PTEN was seen in 11% (15/142) and 17% (24/123) of ERG-positive cases, respectively. PTEN loss (heterozygous or homozygous) was significantly associated with shorter time to biochemical recurrence compared to no PTEN loss (p < 0.001). However, ERG rearrangement versus no rearrangement was not associated with time to PSA recurrence (p = 0.15). Patients who exhibited ERG rearrangement and loss of PTEN had no significant difference in time to recurrence compared to patients with wild-type ERG and loss of PTEN (p = 0.30). Our findings confirm a mutual cooperative role of ERG and PTEN in the pathogenesis of prostate cancer, particularly for homozygous PTEN deletion. ERG did not stratify outcome either alone or in combination with PTEN in this cohort.

Abstract 1945: Degradation of ERG transcription factor as a targeted therapy in B-cell and T-cell acute lymphoblastic leukemia

Abstract Acute lymphoblastic leukemia (ALL) occurs in children and adults and is one of the most common forms of pediatric cancer. The ideal individualized therapy for B- and T-cell ALL heavily depends on the present molecular drivers. However, even with the success of ALL therapy, only a few strategies target disease-specific molecular lesions. Philadelphia chromosome positive (Ph+) ALL, which harbors the BCR:ABL1 rearrangement, are sensitive to tailored inhibitors. Unfortunately, most ALL cases in both children (97-98%) and adults (70-80%) are Ph-; therefore, agents designed to directly target other molecular lesions are urgently needed. ERG, an ETS-family transcription factor, plays a key role in hematopoietic differentiation, megakaryopoiesis, and megakaryoblastic leukemia associated with Down syndrome. ERG overexpression is predictive of a poor prognosis in acute myeloid leukemia and T-ALL. Transgenic expression of ERG causes T-ALL in mice and knockdown of ERG reduces human T-ALL cell proliferation. We have previously identified a consensus peptide (EIPs) that specifically binds to and leads to the proteosomal degradation of the ERG protein in prostate cancers having the TMPRSS2:ERG gene fusion. Here, we demonstrated that wild type ERG was overexpressed at the mRNA and protein levels in a subset of B- and T-cell ALLs, but not in precursor CD4+, CD8+, or CD19+ cells. Pull-down assay found that streptavidin-linked beads were enriched for ERG proteins. Treatment of EIPs induced proteolytic degradation of ERG protein in Hal-01 cells, and therefore selectively inhibited ERG-driven cell growth and proliferation. More importantly, structure-based optimization created the next generation of ERG degraders (EIPv2). EIPv2 treatment significantly decreased ERG protein in Hal-01 cells starting from 3 hrs and nearly depleted ERG proteins at 48 hrs, resulting in 10-fold more potency than EIPs (IC50=2.3µM vs 25.7µM for EIPv2 and EIPs, respectively). Together, our results validate the development of peptidomimetics for ERG depletion as a targeted therapy for ALL. Unlike small-molecule antagonists, which require continuous dosing that eventually induces drug resistance, most protein degraders have relatively higher and longer pharmacological effects without requiring continuous exposure to high doses. Such an approach can abolish the presence of an oncoprotein, which, in theory, can overcome resistance to target inhibitors. Citation Format: Xiaoju Wang, Caleb Cheng, Yuanyuan Qiao, Lanbo Xiao, Cynthia Wang, Abhijit Parolia, Arul Chinnaiyan. Degradation of ERG transcription factor as a targeted therapy in B-cell and T-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1945.

Abstract LB-323: A novel function of ETS transcription factors in alternative splicing is altered in oncogenic FET-ETS fusions

Abstract Ewing sarcomas are characterized by fusions proteins between FET (FUS, EWS and TAF15) family genes encoding RNA binding proteins and ETS (ERG, FLI1 and FEV) family genes encoding transcription factors. FET-ETS fusions regulate both transcription and alternative splicing programs in Ewing sarcoma cells. To date, the function of FET-ETS fusions in splicing is mostly attributed to the FET N-terminal region which is preserved in the fusions. We recently demonstrated that wild type ERG associates with poly-adenylated mRNA and control mRNAs stability in the cytoplasm (Rambout et al., 2016). This therefore indicates that ERG proteins might not be considered only as transcription factors, but can also regulate post-transcriptional processes. On this ground, we hypothesize that ERG proteins might control other post-transcriptional processes, including splicing. In this study, we show that the ERG transcription factor interacts with several spliceosome components and is associated with nascent RNA. Tethered ERG proteins controlled alternative splicing of a minigene reporter in an orientation-specific manner. Deep RNA-seq identified hundreds of alternatively spliced exons regulated by ERG depletion, with an enrichment in RNA motifs bound by RBFOX2 splicing factor. RBFOX2 similarly regulated alternative splicing of common targets and interacted together via the C-terminal domain of ERG which is present in FET-ERG fusions. In Ewing sarcoma cells, RNA-seq identified hundreds of alternatively spliced exons regulated by EWS-FLI1 depletion, with an enrichment in RNA motifs bound by RBFOX2. The oncogenic EWS-FLI1 protein binds RBFOX2 protein but has an antagonist effect on RBFOX2 function. Cross-linking immunoprecipitation (CLIP) experiments show that the EWS-FLI1 fusion inhibits RBFOX2 function by repressing its binding to target pre-mRNAs. We hypothesize that the low complexity prion-like domain of EWS included in the fusion protein may be responsible for sequestration of RBFOX2 outside of the splicing machinery. Importantly, we also show that some of the splicing events induced by the fusion protein may play a critical role in FET-ETS induced cell phenotype. Specific splicing switch partially recapitulate the phenotypic changes that participate in FET-ETS induced cell transformation. These data reveal a new role for ERG family proteins in alternative splicing that is altered in FET-ETS oncogenic proteins, thereby contributing to alternative splicing mis-regulation and cell phenotype in Ewing sarcoma. Citation Format: Olivier Saulnier, Katia Guerdi, Marie-Ming Aynaud, Alina Chakraborty, Josephine Pineau, Christina O Grady, Martin Dutertre, Franck Dequiedt, Olivier Delattre. A novel function of ETS transcription factors in alternative splicing is altered in oncogenic FET-ETS fusions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-323.

The Hematopoietic Transcription Factors RUNX1 and ERG Prevent AML1-ETO Oncogene Overexpression and Onset of the Apoptosis Program in t(8;21) AMLs

The t(8;21) acute myeloid leukemia (AML)-associated oncoprotein AML1-ETO disrupts normal hematopoietic differentiation. Here, we have investigated its effects on the transcriptome and epigenome in t(8,21) patient cells. AML1-ETO binding was found at promoter regions of active genes with high levels of histone acetylation but also at distal elements characterized by low acetylation levels and binding of the hematopoietic transcription factors LYL1 and LMO2. In contrast, ERG, FLI1, TAL1, and RUNX1 bind at all AML1-ETO-occupied regulatory regions, including those of the AML1-ETO gene itself, suggesting their involvement in regulating AML1-ETO expression levels. While expression of AML1-ETO in myeloid differentiated induced pluripotent stem cells (iPSCs) induces leukemic characteristics, overexpression increases cell death. We find that expression of wild-type transcription factors RUNX1 and ERG in AML is required to prevent this oncogene overexpression. Together our results show that the interplay of the epigenome and transcription factors prevents apoptosis in t(8;21) AML cells.

Deregulation of DUX4 and ERG in acute lymphoblastic leukemia.

Chromosomal rearrangements deregulating hematopoietic transcription factors are common in acute lymphoblastic leukemia (ALL). Here we show that deregulation of the homeobox transcription factor gene DUX4 and the ETS transcription factor gene ERG is a hallmark of a subtype of B-progenitor ALL that comprises up to 7% of B-ALL. DUX4 rearrangement and overexpression was present in all cases and was accompanied by transcriptional deregulation of ERG, expression of a novel ERG isoform, ERGalt, and frequent ERG deletion. ERGalt uses a non-canonical first exon whose transcription was initiated by DUX4 binding. ERGalt retains the DNA-binding and transactivation domains of ERG, but it inhibits wild-type ERG transcriptional activity and is transforming. These results illustrate a unique paradigm of transcription factor deregulation in leukemia in which DUX4 deregulation results in loss of function of ERG, either by deletion or induced expression of an isoform that is a dominant-negative inhibitor of wild-type ERG function.

Intragenic ERG Deletions Do Not Explain the Biology of ERG-Related Acute Lymphoblastic Leukemia.

Intragenic ERG deletions occur in 3–5% of B-cell precursor acute lymphoblastic leukemia, specifically in B-other subtype lacking the classifying genetic lesions. They represent the only genetic lesion described so far present in the majority of cases clustering into a subgroup of B-other subtype characterized by a unique gene expression profile, probably sharing a common, however, not yet fully described, biological background. We aimed to elucidate whether ERG deletions could drive the specific biology of this ERG-related leukemia subgroup through expression of aberrant or decreased expression of wild type ERG isoforms. We showed that leukemic cells with endogenous ERG deletion express an aberrant transcript translated into two proteins in transfected cell lines and that one of these proteins colocalizes with wild type ERG. However, we did not confirm expression of the proteins in acute lymphoblastic leukemia cases with endogenous ERG deletion. ERG deletions resulted in significantly lower expression of wild type ERG transcripts compared to B-other cases without ERG deletion. However, cases with subclonal ERG deletion, clustering to the same ERG deletion associated subgroup, presented similar levels of wild type ERG as cases without ERG deletion. In conclusion, our data suggest that neither the expression of aberrant proteins from internally deleted allele nor the reduced expression of wild type ERG seem to provide a plausible explanation of the specific biology of ERG -related leukemia subgroup.

Truncated ERG Oncoproteins from TMPRSS2-ERG Fusions Are Resistant to SPOP-Mediated Proteasome Degradation

SPOP mutations and TMPRSS2-ERG rearrangements occur collectively in up to 65% of human prostate cancers. Although the two events are mutually exclusive, it is unclear whether they are functionally interrelated. Here, we demonstrate that SPOP, functioning as an E3 ubiquitin ligase substrate-binding protein, promotes ubiquitination and proteasome degradation of wild-type ERG by recognizing a degron motif at the N terminus of ERG. Prostate cancer-associated SPOP mutations abrogate the SPOP-mediated degradation function on the ERG oncoprotein. Conversely, the majority of TMPRSS2-ERG fusions encode N-terminal-truncated ERG proteins that are resistant to the SPOP-mediated degradation because of degron impairment. Our findings reveal degradation resistance as a previously uncharacterized mechanism that contributes to elevation of truncated ERG proteins in prostate cancer. They also suggest that overcoming ERG resistance to SPOP-mediated degradation represents a viable strategy for treatment of prostate cancers expressing either mutated SPOP or truncated ERG.

Abstract 456: ERG regulation of intracrine androgen production and castration-resistant prostate cancer progression

Abstract Introduction and Objective: Intratumoral androgen synthesis in prostate cancer (PC) contributes to the development of castration-resistant prostate cancer (CRPC). Several enzymes responsible for androgen biosynthesis have been shown to be overexpressed in CRPC, thus, contributing to CRPC in a castrated environment. However, little is known regarding their mode of regulation and expression in this context. The TMPRSS2-ERG transcription factor has been shown to be present in primary PC tumors as well as CRPC tumors; however, its biological function contributing to the development CRPC in the context of intratumoral androgen synthesis is not known. We hypothesize that TMPRSS2-ERG fusions regulate androgen biosynthetic enzyme (ABE) gene expression and the production of androgens, which contributes to the development of CRPC. Methods: We used a variety of methods including lentiviral knockdown and overexpression of ERG, qPCR, Western blot, ChIP-PCR, and Mass Spectrometry. Results: We found that (a) shRNA knockdown of ERG in VCaP PC cells significantly reduces the expression of ABE's AKR1C3, HSD17B6, and HSD17B4; (b) overexpression of truncated ERG or wild-type ERG in LNCaP PC cells significantly increases the expression of AKR1C3 and HSD17B6; (c) ERG binds to the AKR1C3 gene in VCaP PC cells; and (d) DHT synthesis is reduced in VCaP shERG cells treated with 5α-Androstanedione, a DHT precursor metabolite, compared to VCaP shScrambled controls. Conclusions: These data suggest that AKR1C3 catalyzes the biochemical reduction of 5α-Androstanedione to DHT in PC cells, and that ERG may regulate this step through upregulation of AKR1C3 expression. Elucidation of ERG regulation of ABEs in CRPC may help to stratify TMPRSS2-ERG fusion-positive PC patients in the clinic for anti-hormone driven therapies. Additionally, not all PC tumors express CYP17A1 enzyme, therefore this study emphasizes the importance of investigating ABEs other than CYP17A1, such as AKR1C3 and HSD17B6. In summary, this study addresses alternative ABE drug targets as well as possible drug resistance modes in PC treatment. Citation Format: Katelyn A. Powell, Louie Semaan, Krishna R. Maddipati, Katie (Mary) Conley-Lacomb, Yanfeng Li, Michael Cher, Sreenivasa R. Chinni. ERG regulation of intracrine androgen production and castration-resistant prostate cancer progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 456. doi:10.1158/1538-7445.AM2014-456

Abstract 999: The genomic landscape of pediatric Ewing sarcoma

Abstract The sequencing of aggressive pediatric solid tumors is revealing remarkably stable genomes. In the cases of malignant rhabdoid and retinoblastoma, there is a paucity of recurrently mutated genes, and oncogenesis appears to be driven, at least in part, by epigenetic deregulation. It has been suggested that pediatric tumors characterized by oncogenic fusions will exhibit relatively few additional somatic driver aberrancies. Ewing sarcoma, the second most common pediatric bone tumor, is characterized by rearrangements of the EWS gene and ETS-family transcription factor genes, most commonly FLI and ERG. In experimental models, Ewing sarcoma demonstrates dependency on the expression of the resulting chimeric fusion products. As such, Ewing sarcoma represents a paradigm for studying the genomic landscape of fusion-driven cancers. To this end, we performed whole-exome sequencing of 96 Ewing sarcoma tumors and 11 Ewing sarcoma cell lines, as well as whole-genome sequencing, transcriptome sequencing, and copy-number analysis of a subset of these samples. We found that Ewing sarcoma is one of the most genetically normal cancers sequenced to date, but that treatment, which generally employs genotoxic chemotherapy and radiation, is associated with an increase in mutation rate and single nucleotide substitutions associated with DNA damage. There was a marked absence of recurrent mutations in immediately druggable targets, such as tyrosine kinases, calling into question the feasibility of utilizing tumor sequencing to nominate targeted therapies for patients with Ewing sarcoma. Rather, these results highlight the importance of directly targeting the EWS/ETS fusion events or identifying synthetic lethal dependencies. To this end, we clarified a number of outstanding questions regarding the EWS/ETS fusions. We found that reciprocal ETS/EWS fusions are not expressed in Ewing sarcoma and therefore unlikely to play a role in Ewing pathogenesis as is seen with reciprocal fusions of PML-RARα in acute promyelocytic leukemia. We also found that wild-type FLI and wild-type ERG are not expressed in Ewing sarcoma tumors. However, there appears to be a role for ETS gene deregulation in this disease beyond the expression of EWS/ETS fusion proteins because we found recurrent somatic events in ERF and ETS1. We also identified a small number of other recurrently mutated genes that likely collaborate with EWS/ETS fusions in a minority of cases and confirmed that loss of STAG2 occurs in approximately 15% of Ewing sarcoma tumors. Thus, massively parallel sequencing of a large collection of Ewing sarcoma tumors supports the notion that fusion-driven pediatric malignancies bear quiet genomes, underscores the importance of identifying new treatment approaches targeting EWS/ETS fusions, and also identifies new genetic abnormalities that warrant further biological validation. Citation Format: Brian Crompton, Chip Stewart, Amaro Taylor-Weiner, Gabriela Alexa, Kyle Kurek, Monica Calicchio, Adam Kiezun, Scott Carter, Sachet Shukla, Swapnil Mehta, Aaron Thorner, Carmen de Torres, Cinzia Lavarino, Mariona Sunol, Aaron McKenna, Andrey Sivachenko, Kristian Cibulskis, Michael Lawrence, Lauren Ambrogio, Daniel Auclair, Ivan Rosshandler, Angela Schwarz-Cruz y Celis, Miguel Rivera, Carlos Rodriguez-Galindo, Mark Fleming, Todd Golub, Gad Getz, Jaume Mora, Kimberly Stegmaier. The genomic landscape of pediatric Ewing sarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 999. doi:10.1158/1538-7445.AM2014-999

Abstract 3813: Role of the ERG transcription factor in the resistance of prostate cancer cells to the topoisomerase I inhibitor camptothecin

Abstract Prostate cancer (PCa) is a major public health issue as it is the second cause of death among cancers in industrialized countries, and its incidence is rising. Surgery is the main option for the curative treatment of localized forms of PCa and recurrences are managed with androgen deprivation strategies. In the case of advanced metastatic PCa and when tumors become resistant to castration (mCRPC), chemotherapy with taxanes is initiated. However, response rates remain low with a restricted number of alternatives due to the intrinsic resistance of CRPC to a wide range of cytotoxic agents. Advanced PCa are particularly resistant to topoisomerase I (Top1) inhibitors from the camptothecin (CPT) family, but the mechanistic bases of this resistance have not been investigated in details. Top1 is a nuclear enzyme that induces transient single strand breaks in duplex DNA to ensure the removal of torsional constraints associated with crucial DNA transactions such as replication, transcription, chromosome segregation or DNA recombination. We have previously shown that interaction of Top1 with DNA-PKcs, a kinase involved in non-homologous end-joining, could regulate the cellular response to CPT independently of DNA repair. This was further confirmed by the fact that NU7441, a specific inhibitor of the kinase activity of DNA-PKcs, had no effect on Hela cell sensitivity to CPT. Recently, it was shown that, in PCa cells, DNA-PKcs could interact with ERG, a transcription factor of the ETS family. As advanced PCa are often characterized by a TMPRSS2-ERG fusion leading to wild-type ERG overexpression, we investigated whether ERG could play a role in the resistance of PCa cells to CPT by regulating Top1/DNA-PKcs interaction. We showed that VCaP cells harboring the TMPRSS2-ERG fusion resulting in a 6-fold increase of ERG protein as compared to ERG fusion-negative (control) DU145 or PC3 cells were &amp;gt;100-fold more resistant to CPT. RWPE-1 stably overexpressing low levels of wild-type ERG (2-fold increase) did not show a major change in resistance to CPT as compared to control RWPE-1 cells stably expressing lacZ. Using two specific siRNA, we also showed that transient downregulation of ERG drastically reduced the sensitivity of VCaP cells to CPT, this effect being dependent on the efficacy of ERG repression. Interestingly, this effect was not due to a change in Top1 levels, suggesting that ERG downregulation may affect Top1-DNA-PKcs interaction. We detected this interaction in VCaP cells by immunoprecipitation in the presence of ethidium bromide, indicating that it was independent of DNA and it remains to be seen whether it is disrupted following ERG repression. Our preliminary findings provide evidences for a new mechanism by which ERG and its interaction with DNA-PKcs could explain, at least in part, the intrinsic resistance of PCa to Top1 inhibitors by the regulation of Top1-DNA-PKcs interaction. Citation Format: Emmanuel Roche, Danièle Montaudon, Samer Kayali, Nadine Houédé, Philippe Pourquier. Role of the ERG transcription factor in the resistance of prostate cancer cells to the topoisomerase I inhibitor camptothecin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3813. doi:10.1158/1538-7445.AM2014-3813

ERG and CHD1 heterogeneity in prostate cancer: Use of confocal microscopy in assessment of microscopic foci

Biomarkers predicting tumor response are important to emerging targeted therapeutics. Complimentary methods to assess and understand genetic changes and heterogeneity within only few cancer cells in tissue will be a valuable addition for assessment of tumors such as prostate cancer that often have insufficient tumor for next generation sequencing in a single biopsy core. Using confocal microscopy to identify cell-to-cell relationships in situ, we studied the most common gene rearrangement in prostate cancer (TMPRSS2 and ERG) and the tumor suppressor CHD1 in 56 patients who underwent radical prostatectomy. Wild type ERG was found in 22 of 56 patients; ERG copy number was increased in 10/56, and ERG rearrangements confirmed in 24/56 patients. In 24 patients with ERG rearrangements, the mechanisms of rearrangement were heterogeneous, with deletion in 14/24, a split event in 7/24, and both deletions and split events in the same tumor focus in 3/24 patients. Overall, 14/45 (31.1%) of patients had CHD1 deletion, with the majority of patients with CHD1 deletions (13/14) correlating with ERG-rearrangement negative status (P < 0.001). These results demonstrate the ability of confocal microscopy and FISH to identify the cell-to-cell differences in common gene fusions such as TMPRSS2-ERG that may arise independently within the same tumor focus. These data support the need to study complimentary approaches to assess genetic changes that may stratify therapy based on predicted sensitivities.

Highlights of This Issue

To evaluate whether adherence to dietary recommendations is associated with mortality, George and colleagues examined diets in breast cancer survivors and their association with all-cause and cause-specific mortality. This Women's Health Initiative study included women diagnosed with invasive breast cancer who completed food frequency questionnaires. Women consuming better-quality diets had a 26% lower risk of death from any cause and a 42% lower risk of death from non–breast-cancer causes. Better diet quality was also associated with a reduced risk of all-cause mortality among women with ER+ tumors.Some fertility drugs stimulate ovulation and raise estradiol levels, but their effect on breast cancer risk is unclear. Brinton and colleagues examined the association of ovulation-stimulating drugs and breast cancer. Ever use of clomiphene citrate was not associated with elevated breast cancer risk. However, higher risks were seen for patients who received multiple fertility drug cycles. Since these drugs are primarily used by relatively young women, these findings warrant additional studies.ERG gene rearrangements and PTEN gene deletions occur frequently in prostate cancer. In this study, Fontugne and colleagues evaluated the association of ERG rearrangements and PTEN deletions with oncologic outcomes in prostate cancer patients treated with brachytherapy. Patients with concurrent ERG rearrangement and PTEN deletions demonstrated significantly worse relapse-free survival rates compared to those with wild-type ERG or PTEN. In addition, combined ERG rearrangements and PTEN deletions was independently associated with biochemical recurrence.Although individuals with a strong family history of colorectal cancer (CRC) have a significant risk for CRC, adherence to colonoscopy screening in these groups is low. Lowery and colleagues assessed whether a tailored, telephone counseling intervention can increase adherence to colonoscopy in high-risk families. The authors report that colonoscopy adherence increased 11 percentage points in the tailored, telephone intervention group, compared to no change in a group that received a mailed intervention. In addition, the telephone intervention was associated with a 32% increase in screening adherence compared to the mailed intervention.