EWSR1-FLI1 Fusion Research Articles

Endogenous EWSR1-FLI1 degron alleles enable control of fusion oncoprotein expression in tumor cell lines and xenografts.

Pediatric malignancies frequently harbor chromosomal translocations that induce expression of fusion oncoproteins. The EWSR1-FLI1 fusion oncoprotein acts as a neomorphic transcription factor and is the dominant genetic driver of Ewing's sarcoma. Interrogation of the mechanisms by which EWSR1-FLI1 drives tumorigenesis has been limited by a lack of model systems to precisely and selectively control its expression in patient-derived cell lines and xenografts. Here, we report the generation of a panel of patient-derived EWS cell lines in which inducible protein degrons were engineered into the endogenous EWSR1-FLI1 locus. These alleles enabled rapid and efficient depletion of EWSR1-FLI1. Complete suppression of EWSR1-FLI1 induced a reversible cell cycle arrest at the G1-S checkpoint, and we identified a core set of transcripts downstream of EWSR1-FLI1 across multiple cell lines and degron systems. Additionally, depletion of EWSR1-FLI1 potently suppressed tumor growth in xenograft models validating efforts to directly target EWSR1-FLI1 in Ewing's sarcoma.

Impaired neutrophil-mediated cell death drives Ewing's Sarcoma in the background of Down syndrome.

Ewing Sarcoma (EWS) has been reported in seven children with Down syndrome (DS). To date, a detailed assessment of this solid tumour in DS patients is yet to be made. Here, we characterise a chemo-resistant mediastinal EWS in a 2-year-old DS child, the youngest ever reported case, by exploiting sequencing approaches. The tumour showed a neuroectodermal development driven by the EWSR1-FLI1 fusion. The inherited myeloperoxidase deficiency of the patient caused failure of neutrophil-mediated cell death and promoted genomic instability. In this context, the tumour underwent genome-wide near haploidisation resulting in a massive overexpression of pro-inflammatory cytokines. Recruitment of defective neutrophils fostered rapid evolution of this EWS.

Clinicopathological characteristics and genetic features of young and senior Ewing sarcoma patients

BackgroundEwing sarcoma (EwS) is a highly malignant and heterogeneous tumor. Exploring clinicopathological characteristics and genetic features of EwS is critical for prognosis and treatment regimen.MethodsClinicopathological characteristics and genetic features of young (≤ 30y) and senior (> 30y) EwS patients were analyzed based on histology, phenotype, and next-generation sequencing (NGS) detection.ResultsThe young group (18/36) presented nontypical EwS histological morphology, whereas the senior group (18/36) presented typical morphology. The prognosis of the young group was found to be worse compared with the senior group for patients without metastasis at the initial diagnosis. DNA- and RNA-based NGS was conducted on 20 extraosseous EwS patients. 16/20 samples demonstrated EWSR1-FLI1 fusion and 4/20 demonstrated EWSR1-ERG fusion. However, 13/16 EWSR1-FLI1fusions were detected both in DNA- and RNA-based NGS, 1/16 was detected only at the DNA level, and 2/16 were detected only at the RNA level. An analysis of the genetic profiles of the EWSR1-FLI1 cases revealed that the young group was inclined to couple with more copy number variations (CNV), such as CCND1, CDK4 amplification, and fusion variations, such as CHEK1-EWSR1, SLIT2-EWSR1, and EWSR1-FAM76B fusion. The senior group was more likely to have SNV or Indel mutations, such as EPHA3 and STAG2 mutations. Moreover, patients with more CNV abnormalities had a worse prognosis than those with predominantly SNP variants. In addition, compared with the senior group, the young group had significantly higher CyclinD1 protein expression.ConclusionClinicopathological characteristics and genetic features in young and senior EwS patients differed significantly. Targeting cell cycle dysregulation based on age subgroup may be a potential therapeutic strategy for Ewing sarcoma.

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.

Ganglioside SSEA-4 in Ewing sarcoma marks a tumor cell population with aggressive features and is a potential cell-surface immune target

Carbohydrate markers of immature cells during prenatal human development can be aberrantly expressed in cancers and deserve evaluation as immune targets. A candidate target in Ewing sarcoma is the globo-series ganglioside stage-specific embryonic antigen-4 (SSEA-4). We detected SSEA-4 expression on the cell surface of all of 14 EwS cell lines and in 21 of 31 (68%) primary EwS tumor biopsies. Among paired subpopulations of tumor cells with low versus high SSEA-4 expression, SSEA-4high expression was significantly and consistently associated with functional characteristics of tumor aggressiveness, including higher cell proliferation, colony formation, chemoresistance and propensity to migrate. SSEA-4low versus SSEA-4high expression was not related to expression levels of the EWSR1-FLI1 fusion transcript or markers of epithelial/mesenchymal plasticity. SSEA-4low cells selected from bulk populations regained higher SSEA-4 expression in vitro and during in vivo tumor growth in a murine xenograft model. T cells engineered to express SSEA-4-specific chimeric antigen receptors (CARs) specifically interacted with SSEA-4 positive EwS cells and exerted effective antigen-specific tumor cell lysis in vitro. In conclusion, with its stable expression and functional significance in EwS, SSEA-4 is an attractive therapeutic immune target in this cancer that deserves further evaluation for clinical translation.

Abstract 6254: Casein kinase II (CK2) expression in pediatric solid tumors

Abstract Despite recent advances, high-risk neuroblastoma (NB) and sarcomas account for 15% and 5% of all pediatric cancer deaths, respectively. Many patients attain remission, but approximately half of patients relapse. Genomic alterations such as MYCN amplification and ALK mutations have been shown to be important in prognosis and treatment. Despite improvements, approximately 20% of patients have refractory or recurrent disease after therapy, underscoring the need for further genomic understanding and new approaches to treatment.CK2 is a serine/threonine kinase that regulates numerous cellular processes such as cell growth, proliferation, and survival. Overexpression of CK2 is associated with a poor prognosis in several adult solid tumors, making it a potential target of interest in pediatric solid tumors. This study aims to investigate CK2 expression among patients with NB, Ewing sarcoma (EWS), and rhabdomyosarcoma (RMS) and its correlation with patient survival. MethodsPatients were enrolled in NMTRC008/009: “Feasibility Trial Using Molecular Guided Therapy for Patients with Relapsed/Refractory Childhood Cancer” and BCC-001. WES and RNA-Seq were performed at the Translational Genomics Research Institute and Sema4 or Caris respectively. RNA expression levels were compared to a normal whole-body reference and a pediatric cancer reference. Differential expression data were interpreted in the context of systems biology.ResultsTumor WES was performed on 297 patients (212 NB, 50 EWS, 35 RMS) and RNA-Seq on 284 patients (210 NB, 39 EWS, 35 RMS). The most common mutations or copy number alterations were seen in MYCN, ATRX, ALK, CDKN2A, and chr17q+ in NB; EWSR1-FLI1 fusion in EWS; PAX3-FOXO1 fusion in RMS. RNA expression analysis was performed to understand MYCN-driver status by covariance and interactions and to identify subsets of patients with high/low expression of CK2 in different tumors. CK2 bimodal expression is seen across various cancers and normal tissues, implying distinct sub-groups exist in the data. In NB, overexpression of the CK2 gene with statistical significance was found in MYCN-amplified and chr17q+ tumors. A positive and negative correlation of the CK2 gene is seen with several target genes like MYCN, TRIM71, LIN28B, and HMGA2, JAK2, AR, STAT3, EGFR, LEF1, and NFKB2, respectively. In EWS and RMS, subgroups are identified with high and low expression of the CK2 gene.ConclusionGenomic sequencing of pediatric solid tumors showed a low mutation burden. RNA analysis resulted in the identification of potential novel therapeutic targets involving CK2 pathways. High expression of CK2 correlates with MYCN status. Further analysis of patient molecular subgroups is ongoing. Citation Format: Abhinav Beeravally Nagulapally, Chandrika Behura, Divya Gandra, Giselle Saulnier Sholler. Casein kinase II (CK2) expression in pediatric solid tumors [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 6254.

Abstract 3657: Customized liquid biopsy assay for monitoring EWSR1 fusions in sarcomas

Abstract Background: Gene fusions involving EWSR1 are involved in certain rare sarcomas and sarcoma-like tumors such as, for example, Ewing sarcoma, clear cell sarcoma, hyalinizing clear cell carcinoma, and clear cell odontogenic carcinoma. EWSR1-FLI1 fusions are found in Ewing sarcoma, while EWSR1-ATF1 and EWSR1-CREB are characteristic of clear cell sarcoma among others. Liquid biopsies involve obtaining tumor-derived materials through minimally invasive methods such as blood or other body fluid collection. Analysis of cell-free DNA (cfDNA) is an attractive method to identify gene fusions and monitor tumor recurrence and minimal residual disease (MRD). However, the structural complexity of oncogenic rearrangements poses challenges for the detection using plasma assays. We present a novel assay to monitor fusion positive tumor DNA in a busy oncology clinic. Methods and Patients: Patients with EWSR1 fusion positive sarcomas are identified through clinical RNA-based sequencing. Tumor DNA isolated from biopsy or surgical samples is sequenced to map the intronic breakpoint and customized primers spanning the breakpoint are designed for each patient. The breakpoint is subsequently confirmed through PCR. The product length of ~100bp accounts for the small shedding size of tumor cfDNA. A quantitative TaqMan assay is designed to confirm EWSR1 fusions. The limit of detection is determined by a dilution series of tumor DNA. cfDNA is extracted from blood samples of the patients at multiple timepoints, the tumor burden is quantified by TaqMan Assay and subsequently correlated with clinical findings from imaging studies such as tumor progression or remission. Results: We consented 10 patients with EWSR1 fusion positive tumors to participate in this study. Plasma samples were collected for 8, resulting in 26 total timepoints. Breakpoints were identified in 9 patients and confirmed with PCR on original tumor DNA. TaqMan assays were designed for 5 patients and showed correlation between the tumor burden detected in EWSR1-ATF1 copies in cfDNA extracted from patient blood and the therapeutic response to treatment found through imaging studies. One patient was followed over the period of 9 months, and a total of 5 plasma timepoints were collected. The patient started with a low number of EWSR1-ATF1 copies of <1ng/ul, while on a Iurbinectedin/irinotecan therapy and showed a mixed response in the CT scan. After withdrawing from that therapy and starting other therapy regimens, the EWSR1-ATF1 copies increase to 8ng/ul, correlating with tumor progression observed in CT scans. Conclusion: Our results suggest that ESWR1 fusions in sarcomas can serve as biomarkers to measure tumor burden. Our approach using an individually tailored cfDNA TaqMan assay could provide a valuable monitoring tool to inform clinicians about tumor progression or MRD. With these proof-of-principle findings we can now determine clinical validity and utility. Citation Format: Annie Li, Julia C. Thierauf, Bianca Gonda, Rashi Purohit, Stefan T. Kaluziak, Elizabeth Codd, Stacey N. Dybel, Edwin Choy, Gregory M. Cote, Jochen K. Lennerz, A John Iafrate. Customized liquid biopsy assay for monitoring EWSR1 fusions in sarcomas [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 3657.

Dynamic network curvature analysis of gene expression reveals novel potential therapeutic targets in sarcoma

Network properties account for the complex relationship between genes, making it easier to identify complex patterns in their interactions. In this work, we leveraged these network properties for dual purposes. First, we clustered pediatric sarcoma tumors using network information flow as a similarity metric, computed by the Wasserstein distance. We demonstrate that this approach yields the best concordance with histological subtypes, validated against three state-of-the-art methods. Second, to identify molecular targets that would be missed by more conventional methods of analysis, we applied a novel unsupervised method to cluster gene interactomes represented as networks in pediatric sarcoma. RNA-Seq data were mapped to protein-level interactomes to construct weighted networks that were then subjected to a non-Euclidean, multi-scale geometric approach centered on a discrete notion of curvature. This provides a measure of the functional association among genes in the context of their connectivity. In confirmation of the validity of this method, hierarchical clustering revealed the characteristic EWSR1-FLI1 fusion in Ewing sarcoma. Furthermore, assessing the effects of in silico edge perturbations and simulated gene knockouts as quantified by changes in curvature, we found non-trivial gene associations not previously identified.

PA23 Primary cutaneous Ewing sarcoma in a 14-year-old boy

Abstract A 14-year-old boy presented with a 7-month history of a nodule on the right shoulder. It was initially pink and pea-sized but developed black areas as it grew. It was nontender but bled occasionally. He was otherwise well and took no regular medications. He was originally from the Philippines, having moved to the UK 2 years previously. His mother had a history of breast cancer. This was suspected to be a pyogenic granuloma and treated with a shave biopsy. Histology showed largely patternless sheets of small, blue, round cells, with diffuse and strong membranous reactivity for CD99 on immunohistochemistry. Further molecular analysis revealed EWSR1–FLI1 fusion, confirming Ewing sarcoma. Staging imaging showed no metastatic disease. Following further surgery, he was treated as per the Euro-Ewing protocol with vincristine (V), doxorubicin (D), cyclophosphamide (C), ifosfamide (I), etoposide (E; nine induction cycles: alternate VDC/IE followed by five consolidation cycles IE/VC). Ewing sarcoma most commonly arises from bone and is the second most common bone malignancy in children with an incidence of 2.93 per million. Primary cutaneous Ewing sarcoma is extremely rare, with most of the literature consisting of case reports. A 2015 retrospective review of 56 cases of cutaneous/subcutaneous Ewing sarcoma from the UK and France found that most cases were on the extremities (48%) or trunk (39%). Primary cutaneous Ewing sarcoma tends to occur in late-adolescent or young-adult females, whereas bone disease affects younger patients with a male predominance. Current treatment is similar to treatment for Ewing arising from bone. However, primary cutaneous Ewing sarcoma has a more favourable prognosis, with a 5-year overall survival rate of 93.8%. In summary, cutaneous Ewing sarcoma is a very rare malignancy, particularly in the paediatric population. It is important to consider this in the differential diagnosis of erythematous nodular lesions in children, in order to minimize delays in treatment.

Abstract 4068: Ganglioside SSEA4 in Ewing sarcoma: A marker of tumor cells with highly aggressive features and a potential immune target

Abstract Cancers can aberrantly express carbohydrate antigens restricted to immature cells during prenatal human development, and these may be exploited for selective immune targeting. We studied expression and functional associations of the globo-series ganglioside stage-specific embryonic antigen 4 (SSEA4), a cell surface marker of human embryonic stem cells, in Ewing sarcoma (EwS). Flow cytometry revealed SSEA4 expression on the cell surface of each of 13 EwS cell lines, with moderate to high expression densities in 6 of 13 (46%) cell lines. Of 31 primary EwS tumor biopsies, 21 (68%) were SSEA4 positive by immunofluorescence staining. No associations of SSEA4 positivity with molecular or clinical disease parameters were found. Comparisons of paired tumor cell subpopulations with high and low/negative SSEA4 expression selected by cell sorting from 8 EwS cell lines revealed increased cell proliferation and colony formation of SSEA4high tumor cells, along with higher doxocrubicin chemoresistance and a higher propensity to migrate in transwell assays. SSEA4 negative cells selected from bulk populations of EwS cells regained SSEA4 expression during continued in vitro culture as well as after in vivo xenografting into immunodeficient mice. SSEA4high expression in EwS cells was not dependent on expression levels of the EWSR1-FLI1 fusion transcript or its direct targets, nor associated with markers of epithelial/mesenchymal plasticity. To retarget human T cells to SSEA4, we generated a second-generation, 41BBζ CAR using the antigen-binding domains of a murine anti-SSEA4 monoclonal antibody. SSEA4-specific CAR T cells specifically interacted with SSEA4 positive EwS cells in a strictly antigen-dependent manner, resulting in effective tumor cell lysis in vitro. We conclude that targeting of SSEA4 with CAR T cells or alternative immune therapeutics could be an attractive strategy to eliminate tumor cell subsets with high propensity to drive disease progression in EwS and therefore deserves further evaluation towards clinical translation. Citation Format: Silke Jamitzky, Bianca Altvater, Carolin Krekeler, Laura Hoen, Caroline Brandes, Lisa Richter, Julia Ebbinghaus, Laurin Ochs, Nicole Farwick, Katja Urban, Dennis Görlich, Ian Johnston, Rita Pfeifer, Claudia Rossig, Wolfgang Hartmann, Sareetha Kailayangiri. Ganglioside SSEA4 in Ewing sarcoma: A marker of tumor cells with highly aggressive features and a potential immune target. [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 4068.

Primary Cutaneous Ewing Sarcoma of the Scalp With Metastasis to the Lung: An Unusual Manifestation During Pregnancy

A 32-year-old G2P1L1 (5 months pregnant) woman presented with a 3-month history of a slow-growing cystic lesion on her scalp vertex. Similar lesions in the exact location were excised twice in the past with a diagnosis of trichilemmal carcinoma (TC). A biopsy of the scalp lesion showed morphology and immunoprofile consistent with previously diagnosed TC. Staging PET/CT demonstrated a 4.7 cm right upper lobe lung, and a subsequent lung biopsy showed a small, round blue-cell tumor with necrosis, morphologically identical to the prior biopsies from the scalp. Considering the unusual clinical course of TC, a lung biopsy was sent for next-generation sequencing that showed EWSR1-FLI1 (type1) fusion. Additionally, CD99 immunostaining revealed uniform cytoplasmic and membranous staining in the tumor cells. The previous scalp excision specimen was also sent for mutation analysis, which showed EWSR1-FLI1 fusion. In conjunction with clinical history and histological and molecular findings, a definitive diagnosis of primary cutaneous Ewing sarcoma (PCES) with local recurrence and metastasis to the lung was made. We present a case of PCES, which was previously misdiagnosed and treated as TC. This case emphasizes the importance of CD99 in the initial screening of cutaneous small round blue-cell tumors to avoid misdiagnosis from other morphological overlaps. Also, despite its rarity, PCES should be included in the differential diagnosis of small, round, blue cell tumors at cutaneous sites. Our case also exemplifies common biases in medical decision-making, including premature closure and anchoring bias which can result in misdiagnosis or diagnostic delay and associated delay in appropriate management.

Abstract PR009: A new invasive zebrafish model of ewing sarcoma reveals EWSR1-FLI1-driven dysregulation of heparan sulfate proteoglycan metabolism and ERK signaling in developing tumors

Abstract Ewing sarcoma is a malignant tumor of bones or soft tissues with an extremely poor prognosis for patients with metastatic or relapsed disease. Ewing sarcoma is associated with the appearance of a chimeric fusion oncogene, most frequently EWSR1-FLI1 (EF1). The interaction of Ewing sarcoma cells with the tumor microenvironment is essential for tumor growth, angiogenesis, and metastasis. The tumor microenvironment includes surrounding blood vessels, immune cells, signaling molecules, and extracellular matrix. The dependence of a tumor cell on its microenvironment opens new possibilities to target not just cancer cells but also the tumor microenvironment itself. In the absence of a representative in vivo model, the role of tumor cell/microenvironmental interactions in Ewing sarcoma initiation and progression is not well-understood. To address these concerns, we developed a new in vivo genetic model of Ewing sarcoma by integrating the human EWSR1-FLI1 fusion oncogene into the zebrafish genome. Our system allows broad mosaic Cre-inducible expression of human EF1 in zebrafish, which causes rapid onset of Ewing sarcoma at high penetrance. This invasive model allows studying the behavior of GFP-labeled cancer cells during tumor initiation and progression, in a complex developmental context which is not currently possible in mammals. We found that observed tumors express canonical EF1 target genes and stain for known Ewing sarcoma markers including CD99. Proteomic analysis revealed that EF1 expression affects the expression of enzymes involved in heparan sulfate proteoglycan (HSPG) metabolism. Such dysregulation of proteoglycan metabolism is associated with activation of ERK1/2 signaling pathway in tumor cells resulting in increased tumor cell proliferation and survival. Proteoglycans are key regulators of signaling of cell surface receptors, playing an essential role in cell-to-cell communication. Targeting heparan sulfate proteoglycans with the specific heparan sulfate antagonist Surfen reduces ERK1/2 signaling and decreases tumorigenicity of Ewing sarcoma cells in vitro and in vivo. Taken together, our new zebrafish model will open new possibilities to study EWSR1-FLI- driven tumorigenesis, as well as mechanisms of tumor development and progression. Our results highlight the important role of the extracellular matrix in Ewing sarcoma tumor growth and the potential of agents targeting proteoglycan metabolism as novel therapies for this disease. Citation Format: Elena Vasileva, Mikako Warren, Timothy J. Triche, James F. Amatrud. A new invasive zebrafish model of ewing sarcoma reveals EWSR1-FLI1-driven dysregulation of heparan sulfate proteoglycan metabolism and ERK signaling in developing tumors [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr PR009.

Validation of PRKCB Immunohistochemistry as a Biomarker for the Diagnosis of Ewing Sarcoma

Background: Ewing sarcoma (ES) can be confirmed by identifying the EWSR1-FLI1 fusion transcript. This study is to investigate whether immunostaining (IHC) of PRKCB—a protein directly regulated by EWSR1-FLI1 is a surrogate maker for diagnosing ES in routine practice. Methods: Microarray gene expression analyses were conducted. RKCB IHC was applied to 69 ES confirmed by morphology and molecular methods, and 41 non-Ewing small round cell tumors. EWSR1 rearrangement, EWSR1-FLI1 fusion or t(11;22)(q24;q12) were identified by fluorescence in situ hybridization, reverse transcriptase polymerase chain reaction, or cytogenetic analysis, respectively. Results: Gene array analyses showed significant overexpression of the PRKCB in ES. PRKCB IHC was positive in 19 cases of ES with EWSR1-FLI1 fusion, 3 cases with cytogenetic 11:22 translocation and 59 cases with EWSR1 rearrangement while negative in only one EWSR1 rearranged case. PRKCB IHC is sensitive (98%) and specific (96%) in detecting EWSR1 rearranged ES. Conclusions: PRKCB is a reliable antibody for diagnosing ES in routine practice.

TK216 targets microtubules in Ewing sarcoma cells

SummaryEwing sarcoma (EWS) is a pediatric malignancy driven by the EWSR1-FLI1 fusion protein formed by the chromosomal translocation t(11; 22). The small molecule TK216 was developed as a first-in-class direct EWSR1-FLI1 inhibitor and is in phase II clinical trials in combination with vincristine for patients with EWS. However, TK216 exhibits anti-cancer activity against cancer cell lines and xenografts that do not express EWSR1-FLI1, and the mechanism underlying cytotoxicity remains unresolved. We apply a forward-genetics screening platform utilizing engineered hypermutation in EWS cell lines and identify recurrent mutations in TUBA1B, encoding ⍺-tubulin, that prove sufficient to drive resistance to TK216. Using reconstituted microtubule (MT) polymerization in vitro and cell-based chemical probe competition assays, we demonstrate that TK216 acts as an MT destabilizing agent. This work defines the mechanism of cytotoxicity of TK216, explains the synergy observed with vincristine, and calls for a reexamination of ongoing clinical trials with TK216.

Abstract 80: Genomic characterization of PDX models from rare cancer patients in the NCI Patient-Derived Models Repository

Abstract Background: The National Cancer Institute’s Patient-Derived Models Repository (NCI PDMR; https://pdmr.cancer.gov) has developed a large number of patient-derived xenograft (PDX) models from a diverse set of rare cancers. These models have been genomically characterized using whole-exome sequencing (WES) and RNAseq. The resource provides a unique opportunity to explore the genomic features of rare tumor models in NCI PDMR and to understand the oncogenic processes in pre-clinical models to identify biomarkers associated with therapeutic responses. Methods: Genomic characterization was done in 4-6 PDX samples across multiple passages and lineages from each model. As the samples exhibited a high level of genomic stability within each model, consensus mutation and copy number variation (CNV), microsatellite instability (MSI), genomic loss of heterozygosity (LOH), homologous recombination deficiency score (scarHRD), and mutational signature data were generated from WES. Fusions were identified from RNASeq data using Star-Fusion and FusionInspector. Gene set enrichment analysis was conducted from the gene expression data obtained from RNAseq. Results: 1) 233 PDX models have been developed and characterized from more than 45 different rare malignancies. Most frequent cancer types are different sarcomas (n=63), head & neck squamous cell carcinoma (n=61), and malignant fibrous histiocytoma (MFH) (n=11); 2) TP53 was the most frequently altered gene, mutated in 51% of models, followed by NOTCH1 (16%) and PIK3CA (11%). In terms of CNVs, ovarian epithelial cancer (OVT) showed relatively high chromosomal instability, while uterine endometrioid carcinoma (UEC) and synovial sarcoma (SYNS) had low instability; 3) MSI-H was observed in only 7 models. Esophageal adenocarcinoma (ESCA), OVT, and cervical squamous cell carcinoma (CESC) had high scarHRD and genomic LOH scores, while both scores were low in UEC and anal squamous cell carcinoma (ANSC). COSMIC v2 mutational signature 3 is significantly associated with a high scarHRD score (p-value < 0.01, Wilcoxon rank-sum test); 4) Characteristic fusions were observed in certain sarcoma models: SS18-SSX1 and ASPSCR1-TFE3 fusions were observed in SYNS and alveolar soft part sarcoma (ASPS) models respectively. EWSR1-FLI1 fusion was present in 2 out of 3 Ewing sarcoma (ES) models. 5) Gene set enrichment analysis from RNASeq data showed that epithelial-mesenchymal transition score could accurately distinguish carcinoma from sarcoma models, confirming the divergent gene expression programs. Conclusion: Comprehensive genomic characterization of NCI PDMR models generated from rare cancers solves an unmet need in the community. It will serve as a valuable resource for translational researchers interested in pre-clinical drug development and discovery. Citation Format: Li Chen, Rini Pauly, Ting-Chia Chang, Biswajit Das, Yvonne A. Evrard, Chris A. Karlovich, Tomas Vilimas, Alyssa Chapman, Nikitha Nair, Luis Romero, Anna Lee Fong, Amanda Peach, Shahanawaz Jiwani, Nastaran Neishaboori, Lindsay Dutko, Kelly Benauer, Gloryvee Rivera, Erin Cantu, Corinne Camalier, Thomas Forbes, Michelle Gottholm-Ahalt, John Carter, Suzanne Borgel, Chelsea McGlynn, Candace Mallow, Emily Delaney, Tiffanie Miner, Michelle A. Eugeni, Dianne Newton, Melinda G. Hollingshead, P. Mickey Williams, James H. Doroshow. Genomic characterization of PDX models from rare cancer patients in the NCI Patient-Derived Models Repository [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 80.

Abstract 902: Identification, characterization, and targeting of desmoplastic small round cell tumor cancer stem cell-like cells

Abstract Desmoplastic Small Round Cell Tumor (DSRCT) is a rare and aggressive form of pediatric cancer caused by a translocation between chromosomes 11 and 22, creating the novel EWSR1-WT1 fusion gene. Current therapy including surgery and the P6 chemotherapy regimen is insufficient, leading to a 5-year survival rate of only 15-25%. One potential explanation for this poor prognosis is the existence of a cancer stem cell (CSC) population with the ability to resist chemotherapy and cause both tumor recurrence and metastasis. While a CSC population has been identified in the closely related Ewing Sarcoma, most commonly caused by the EWSR1-FLI1 fusion gene, no CSC population has yet been identified in DSRCT. In this study, novel culture conditions were developed that enabled the formation of tumorspheres in vitro in the two commonly available DSRCT cell lines: JN-DSRCT-1 and BER-DSRCT. These novel culture conditions led to upregulation of stemness genes at the RNA and protein level including NANOG and SOX2, two genes that are associated with poor survival outcomes in TCGA sarcomas data. CCK-8 assay demonstrated an increased chemoresistance for tumorspheres versus normal adherent culture especially for doxorubicin, which was confirmed by reduction in PARP cleavage. This chemoresistance may be partially explained by a more quiescent CSC state as shown by reduced cell proliferation and a lower percentage of cells in the S and G2/M phases. However, cells grown in tumorspheres still robustly formed tumors in vivo, thus demonstrating the two hallmark CSC traits of chemoresistance and tumor formation. RNA-seq was performed to elucidate key differences between the adherent and sphere culture conditions and identify potential targets for the therapeutically important CSC-like population. Overrepresentation analysis and gene set enrichment analysis both showed upregulation of pathways related to chromatin assembly and disassembly, suggesting epigenetic changes as DSRCT cells move to a more stem-like state. RNA-seq further revealed kinases upregulated in tumorspheres including B-lymphocyte kinase (BLK) which previous studies have demonstrated is oncogenic, regulated by EWSR1-WT1, and not necessary for normal survival. BLK knockdown reduced CSC-like properties including abrogation of tumorsphere formation and reduction in the levels of SOX2 and NANOG. Together, this work for the first time identifies a CSC-like population in DSRCT and BLK as a potential DSRCT CSC target. Citation Format: Justin W. Magrath, Alifiani B. Hartono, Sean B. Lee. Identification, characterization, and targeting of desmoplastic small round cell tumor cancer stem cell-like cells [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 902.

Abstract 2299: Function of Ewing sarcoma EWSR1 protein in the maintenance of chromosome stability

Abstract Ewing sarcoma is the second most common bone cancer that develops in adolescents and young adults. Majority of Ewing sarcoma patients (~85%) carry an EWSR1-FLI1 fusion gene derived from t(11;22)(q24;q12) chromosomal translocation. The formation of EWSR1-FLI1 leads to the loss of one wild type EWSR1 allele, however, how loss of the gene contributes to the pathogenesis of Ewing sarcoma remains unclear. Our previous study revealed that the EWSR1 is required for a faithful mitosis. We demonstrated that the mutant ewsa (a homologue of human EWSR1) zebrafish induces mitotic defects, aneuploidy, and promotes tumorigenesis (with tp53 mutation). Consistently, Ewing sarcoma patients display high incidence of aneuploidy (e.g. approximately 47% of the patients display trisomy 8). For these reasons, we aim to elucidate the molecular function of EWSR1 in faithful chromosome segregation and in maintenance of chromosome stability. To accomplish our goal, we employed the Auxin-Inducible Degron (AID) system because it allows us to conditionally degrade EWSR1 proteins in a cell line. Both EWSR1 alleles were tagged with mini-AID using CRISPR/Cas9 system in a stable DLD-1 cell that expresses a plant E3 ligase TIR1. The western blot verified the efficient degradation of EWSR1 when the cells were treated with Auxin (AUX+) for 24hrs. To determine whether EWSR1 maintains chromosomal stability, the cells were synchronized to pro-metaphase using thymidine/nocodazole protocol, and were treated with AUX for 24hrs (underwent one cell cycle) concurrently. The EWSR1 knockdown (AUX+) cells displayed significantly higher incidence of lagging chromosomes compared to the control (AUX-) cells. Furthermore, immunocytochemistry using the antibody against the key mitotic regulator, Aurora B, revealed that the EWSR1 knockdown (AUX+) cells displayed significantly less incidence of Aurora B localization at inner centromere, but higher incidence of localization on kinetochore compared to the control (AUX-) cells. Consistently, when the cells that were treated with AUX for 48hrs (underwent two cell cycles), it displayed significantly higher incidence of aberrant numbers of chromosome compared to the control (AUX-) cells. In general, impaired chromosome segregation leads to mitotic delay, accompanied by the activation of mitotic checkpoint. However, the EWSR1 knockdown (AUX+) cells did not undergo any arrest in mitosis. Therefore, our data suggest that the EWSR1 knockdown (AUX+) cells not only induces aneuploidy by inducing lagging chromosome, but also it may override mitotic checkpoint. Currently, we are in a process of elucidating the molecular mechanism of EWSR1 knockdown dependent mitigation of mitotic arrest. Successful completion of the study may shed light on how/whether haploinsufficiency of EWSR1 contributes to the molecular pathogenesis of Ewing sarcoma. Citation Format: Haeyoung Kim, Mizuki Azuma. Function of Ewing sarcoma EWSR1 protein in the maintenance of chromosome stability [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 2299.

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

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

Genomic co-genotype classification and clinical prognosis in Ewing sarcoma.

e23532 Background: Ewing sarcoma is a highly aggressive round cell mesenchymal tumor that commonly occurs in children and young adults. It is molecularly characterized by chromosomal translocations, most commonly t (11;22), resulting in the abnormal EWSR1-FLI1 fusion. In addition to EWSR1-FLI1, patients may have other genomic variants that can be used for risk stratification. In the present study, we retrospectively investigated the association between co-variant genes and prognosis in patients with Ewing sarcoma. Methods: From 2019 to 2021, 10 patients with Ewing sarcoma were enrolled in the study, and their standard first-line treatment consisted of five-drug regimen chemotherapy including vincristine, doxorubicin (Adriamycin) and cyclophosphamide, alternating with ifosfamide and etoposide (VAC/IE). We carried out whole-exome sequencing on collected tumor samples and matched leukocyte DNA from these patients. Mutations, indels, gene deletions, and amplifications were identified, and survival analysis was performed. Results: In general, median progression-free survival (PFS) of the 10 confirmed Ewing sarcoma (with EWSR1-FLI1 fusion) patients was 10.5 months. We found that 60%, 30%, and 30% of patients had genetic variants (including mutation and copy number variation) in the three signaling pathways of cell cycle, WNT, and NOTCH, respectively. Their PFS was shorter than patients without these variants (log-rank test, p = 0.0345, 0.0053, and 0.0053, respectively). Median PFS was 7 vs 17 months in cell cycle pathway (the former with variants and the latter without variants), 5 vs 14 months in WNT pathway, and 5 vs 14 months in NOTCH pathway. However, there was no difference in PFS between patients with and without variants in other pathways (including TP53, RTK-RAS, MYC, DDR, PI3K) (log-rank test, p＞0.05). Conclusions: We found that the variants in cell cycle, WNT and NOTCH signaling pathway were associated with poor prognosis of Ewing sarcoma. This study provides important molecular markers for risk stratification in patients with Ewing sarcoma, which may provide insights into later clinical trials.

Establishment and characterization of a novel patient-derived Ewing sarcoma cell line, NCC-ES2-C1.

Ewing sarcoma (ES) is a small round cell sarcoma that is characterized by the unique gene translocation EWSR1-FLI1. It is the second most common primary bone and soft tissue malignancy in children and adolescents. It constitutes 10-15% of all bone sarcomas and is highly aggressive and rapidly recurring. Although intensive treatments have improved the clinical outcome of ES patients, 20-25% of them exhibit metastases during diagnosis. Thus, the prognoses of these patients remain poor. Celllines are pivotal resources to investigate the molecular background of disease progression and to develop novel therapeutic modalities. In this study, we establishedand characterized a novel ES cell line, NCC-ES2-C1. The presence of the EWSR1-FLI1 fusion gene in these cells was confirmed in the NCC-ES2-C1 cells. Furthermore, these cells exhibited constant proliferation, and invasion, but did not form tumors in mice. We screened the anti-tumor effects of 214 anti-cancer drugs in NCC-ES2-C1 cells and found that the drugs which effectively reduced the proliferation of NCC-ES2-C1 cells. We concluded that NCC-ES2-C1 cells are a useful resource to study functions of the EWSR1-FLI1 fusion gene, investigate phenotypic changes caused by genes and proteins, and evaluate the anti-tumor effects of novel drugs.