Abstract Introduction: Ewing sarcoma (ES) is an aggressive pediatric bone and soft tissue cancer that is absolutely dependent on the EWS::FLI1 fusion transcription factor. Despite this dependency, ES tumors demonstrate substantial variability in EWS::FLI1 transcriptional activity both across tumors and within individual tumors. Here, we investigate the inter- and intra-tumoral heterogeneity of EWS::FLI1 and its functional importance. Methods: We established a highly optimized siRNA knockdown protocol for EWS::FLI1 in 6 preclinical ES cell line models to achieve equal levels of suppression and evaluate differences in induced and repressed transcriptional targets and DNA binding events of EWS::FLI1. In order to investigate underlying mechanisms driving resistance, we employed state-of-the-art techniques including Cleavage Under Target (CUT&Tag), single nuclei RNA sequencing, and spatial transcriptomics of preclinical cell line models and ES patient samples to characterize the inter- and intra-tumoral heterogeneity of EWS::FLI1. Results: Differential expression analysis of 6 cell lines with knockdown of EWS::FLI1, revealed that each cell line exhibited as many unique induced targets as shared ones, and repressed targets were even more cell line specific. The heterogeneity was functionally important as cell migration and migration signatures varied among the 6 models. Knockdown of EWS::FLI1 resulted in increased migration in one cell line and impaired migration in other cell lines. Intratumoral heterogeneity was also evident in single cell data with multiple transcriptional clusters evident in different proportions across the models. These different clusters also appear to be functionally important as at least two clusters resisted suppression by the EWS::FLI1 targeted agent, trabectedin, in patient samples collected on a recent trial. The heterogeneity was rooted in a combination of cell line specific EWS::FLI1 DNA binding events determined by CUT&Tag, cell specific copy number variants (CNV), and cofactor expression. Conclusion: Our data suggests that EWS::FLI1 exhibits highly unique transcriptional activity between models which leads to a heterogeneous representation of target genes. In patients, EWS::FLI1 heterogeneity leads to the expression of a high-risk population of cells resistant to EWS::FLI1 targeting. We are working to understand mechanisms driving the high-risk population of ES cells and identify new druggable targets for these transcriptionally distinct tumor cell populations. Citation Format: Rachael Hinshaw, Zachary P. Tolstyka, Susan M. Kitchen-Goosen, Seneca Kinn-Gurzo, Rebecca Kaufman, Maggie Chasse, Elizabeth Wilson, Gretchen Lam, Stephanie The, Elissa Boguslawski, Patrick J. Grohar. Defining the intertumoral and intratumoral transcriptional heterogeneity of EWS::FLI1 in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 635.

Abstract Osteosarcoma (OS) is a bone tumor that affects human and canine patients. Standard of care is neoadjuvant chemotherapy and surgery resulting in a 5 year survival rate for patients with localized disease of ∼70%. However, patients with metastatic disease and relapsed disease have a 5 year overall survival of less than 30%. Therefore, there is a critical need for improved therapies and a better understanding of the biological underpinnings of high risk disease. A subset of patients with particularly poor outcomes are known to have copy number amplification of MYC. However, it is not known if MYC contributes to the high risk phenotype by driving metastatic progression or drug resistance. Importantly, 20 compounds have been described as MYC inhibitors and perturb different steps of MYC driven transcription. In this report, we found that MYC drives cell migration and outgrowth but does not appear to contribute to drug resistance in OS cells. More precisely, MYC silencing reversed the metastatic phenotypes of migration and outgrowth of OS cells. Further, MYC downstream targets play an important role in metastatic progression. Silencing of MYC in 5 different cell lines revealed 45 common induced targets, many of which are known to modulate different steps in the metastatic cascade. We screened all 20 compounds previously shown to interfere with MYC transcription using an approach designed to capture the compound that modulates both MYC activity and the metastatic phenotype. Fourteen compounds modulated expression of MYC and/or downstream targets in 4 different OS models. Of those,10 compounds had a profound impact in cell viability in both 2D and 3D assays. Five of these showed selective toxicity in 3D relative to 2D; a phenotype linked to metastatic progression. Importantly, not all compounds that modulated MYC showed therapeutically favorable effects on migration or metastatic organization and outgrowth with at least 2 compounds driving a dramatic increase in migration despite suppressing expression of MYC. Nevertheless, 2 compounds, samuraciclib and THZ531, blocked MYC expression, downstream target expression, cell migration, metastatic organization and outgrowth. We confirmed these results and showed reversal of metastatic competence and complete reversal of metastatic outgrowth using the in vivo/ex vivo pulmonary metastasis assay (PuMA). We are now working to integrate CUT&Tag with BRUseq, an assay of nascent transcription, to determine if modulation of different steps in MYC transcription drives diverse cellular phenotypes as we hypothesized. Nevertheless, the top hit of the screen, samuraciclib, convincingly reverses MYC activity and the associated metastatic phenotype and is undergoing additional testing in metastatic OS mouse models and a canine clinical trial is under development. Correlative biology such as spatial transcriptomics will be used to guide the translation of samuraciclib to patients with high risk osteosarcoma. Citation Format: Emily Seiden, Scott Sauer, Emma Hiscock, Nha Nhu Le, Ainsley Hellens, Monica Inda, Andrew Fuller, Sridhar M. Veluvolu, Rachael Hinshaw, Zachary P. Tolstyka, Elissa Levine, Rashmi Chugh, Theodore W. Laetsch, Nouri Neamati, Heather Wilson-Robles, Chand Khanna, David Warshawsky, Patrick J. Grohar. Targeting high risk osteosarcoma: MYC modulation alters metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1151.

TPS10057 Background: Ewing sarcoma (EWS) is an aggressive tumor of the bone and soft tissue primarily affecting adolescents and young adults. For patients with recurrent disease, outcomes remain poor with 5-year survival rates of 10%–15%, highlighting the need for novel approaches. STEAP1 is highly expressed in EWS cell lines, xenograft models, and patient tumor samples 1 and is transcriptionally regulated by EWSR1::FLI1 , the main oncogenic driver in EWS. Xaluritamig, a humanized bispecific XmAb 2+1 T-cell engager targeting STEAP1 and CD3, has demonstrated potent, antigen-dependent T-cell–mediated lysis of STEAP1-expressing EWS cell lines 1 and induced robust in vivo antitumor activity in EWS xenograft models with concomitant CD8+ T-cell activation. In metastatic castration-resistant prostate cancer, another STEAP1-expressing solid malignancy, xaluritamig has shown meaningful antitumor activity and a manageable safety profile 2 supporting its clinical evaluation in R/R EWS. Methods: This ongoing, phase 1b, single-arm, open-label, two-part, multicenter study (NCT07297979) is evaluating xaluritamig in pediatric, adolescent, and adult patients with a histologic diagnosis of EWS and an EWSR1 :: ETS fusion gene locally confirmed by next-generation sequencing. Patients must have R/R EWS following ≥1 line of systemic therapy and Karnofsky or Lansky performance status ≥70. Part 1 (dose confirmation) consists of cohort 1 (age ≥12 years) and cohort 2 (age ≥2 to <12 years), and part 2 (dose expansion) includes patients aged ≥2 years with no upper age limit at enrollment. This study includes a 21-day screening period, a treatment period, a safety follow-up period, and a long-term follow-up period. Patients receive xaluritamig every 2 weeks once reaching the target dose until radiographic disease progression per RECIST v1.1, clinical disease progression, unacceptable toxicity, initiation of other anticancer therapy not permitted per protocol, withdrawal of consent, death, or end of study as determined by the sponsor, whichever is earlier. Primary endpoints are safety, tolerability, and determination of recommended dose(s) for expansion. Secondary endpoints are pharmacokinetics, preliminary antitumor activity, and immunogenicity. Biomarker evaluation as an exploratory endpoint includes assessment of serum cytokines, other pro-inflammatory markers, immune-cell subsets, circulating tumor DNA, STEAP1 protein expression, and tumor genomic profile and their association with safety and efficacy. CRS mitigation plan includes step-up dosing, steroid premedication, and administration of interleukin-6 inhibitors as needed. Site activation commenced in January 2026 and enrollment is ongoing. 1. Nolan-Stevaux O. Cancer Res. 2020;80(16_Suppl):DDT02–03. 2. Kelly WK, Danila DC, Lin CC, et al. Cancer Discov . 2024;14(1):76-89. Clinical trial information: NCT07297979 .

Abstract Introduction: Ewing sarcoma (EwS) is a FET::ETS family member-driven primary bone cancer demonstrating vast heterogeneity. Between a patient’s primary diagnosis and disease progression, tumor cell state adaptation, microenvironment changes, and the landscape of evolving therapeutic vulnerabilities remain poorly understood. To address these gaps, the Sean Karl Cohort was established in 2025 to conduct the largest single-cell transcriptomic analysis of retrospective paired tumor samples from patients with EwS. Here, we present data from the five analytic teams, and through shared SOPs now expand this cohort to Europe. Methods: Common sample processing SOPs were established to isolate cells from FFPE material from paired patient EwS samples. Single-cell (sc) RNAseq was generated using the GEM-X Flex Gene Expression protocol (10x Genomics). Alex’s Lemonade Stand Foundation (ALSF) Data Lab established a common data processing and integration pipeline with an EwS-specific cell annotation workflow to create a harmonized dataset for downstream analyses. Analytic teams were created to define tumor cell subpopulations and their therapeutic vulnerabilities and to characterize the tumor microenvironment. Data: We demonstrate the feasibility of generating high-quality scRNAseq profiles from retrospective FFPE-preserved EwS tumors. To date, 116 samples have yielded ∼800,000 cells. Integrated analysis reveals reproducible EwS tumor cell states shared across patients with regulatory network analyses nominating candidate therapeutic vulnerabilities. The scale of this international cohort enables saturation analysis for rare cell populations. Paired and longitudinal samples further allow correlation of emergent cell states with therapy resistance and metastatic progression. For example, immune-focused analyses show increases in T cell and macrophage populations in post-therapy samples. A novel, standardized EwS-specific cell annotation workflow has been developed to harmonize analyses and findings. These data will be openly shared with the community through the ALSF Single-cell Pediatric Cancer Atlas Portal. Conclusion: Large-scale international collaborative sample sharing, standardized processing pipelines, and harmonized EwS-specific annotations now enable deep single-cell analyses to characterize tumor heterogeneity in this rare pediatric and adolescent cancer. An in vivo expansion of the Sean Karl Cohort is underway to assess conservation of EwS tumor cell states and regulatory vulnerabilities identified in human tumors pre- and post-therapy in preclinical models, further supporting translation of these findings toward therapeutic strategies. Citation Format: Abbe Pannucci, Elina Mukherjee, Jessica D. Daley, Shireen Sita Ganapathi, Elissa Boguslawski, Lea Surrey, Lauren Gutstein, Patrick Azar, Emily Stockfisch, Azfar Neyaz, Ivy John, Jennifer Picarsic, Yutaro Tanaka, Byron Butaney, Riaz Gillani, Brian D. Crompton, Katherine A. Janeway, Jaclyln Taroni, Jessica Davis, Damon Reed, Adam Shlien, Theodore W. Laetsch, Rajen Mody, Clémence Henon, Thomas G. Grünewald, Elizabeth R. Lawlor, Filemon Dela Cruz, Patrick J. Grohar, Jovana Pavisic, Ally Hawkins, Anthony R. Cillo, Kelly M. Bailey. The Sean Karl Cohort: An international single-cell RNAseq study of paired patient Ewing sarcoma specimens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 643.

Abstract Background: We recently defined the NB/EWS surfaceomes using integrative proteogenomics to prioritize proteins as candidate immunotherapeutic targets. GFRA2 was a top ranked candidate for both NB and EWS (Clin Cancer Res 2023, Cancer Cell 2024). Aims: (1) Validate and assess mechanism of GFRA2 overexpression; (2) Identify selective antibody binders to GFRA2; (3) Engineer antibody drug conjugates (ADCs) and test for internalization and potency. Methods: ChIP- and RNA-sequencing of NB and RNA-sequencing following siRNA depletion of EWSR1::FLI1 were used to evaluate mechanisms of GFRA2 overexpression. GFRA2 abundance and cellular localization was evaluated by flow cytometry and immunofluorescence. Phage display was performed with recombinant GFRA2 extracellular domain protein as the bait and the highly conserved GFRA1 and GFRA3 recombinant proteins as counters. In parallel, we humanized the anti-GFRA2 murine HSAN antibody. While we plan on reporting on several linker-payload combinations, here we focus on the initial humanized HSAN antibody conjugated to pyrrolobenzodiazepine (PBD) via a cleavable linker, which was tested for internalization using live cell imaging and cytotoxicity across a panel of human NB and EWS preclinical models. Results: We identified a proximal super enhancer (Percentile: 97.4%-99.7%) or enhancer (94.4%-97.7%) in all 10 NB cell lines profiled. Depletion of EWSR1::FLI1 resulted in significantly decreased GFRA2 mRNA expression across 6 EWS cell lines (P<0.0001), and this was validated via immunoblotting. GFRA2 protein was expressed uniformly and to variable degrees on the cell surface of 10 NB cell lines, 8 NB patient-derived xenografts, and 6 EWS cell lines by flow cytometry and/or immunofluorescence. Patient tumor RNA-Seq showed 92.8% of NB (N=126; median=47.65) and 74% of EWS (N=85, median=11.47) had high expression defined as a GFRA2 TPM > 5. Phage display panning of fully human antibody fragments yielded three Fabs and one VH single domain antibody specific to GFRA2 with moderate binding affinity (50-500 nM). Humanized HSAN (co-hu-HSAN) showed high binding affinity specific to GFRA2 (1-10 nM). We then showed robust internalization of co-hu-HSAN across 6 NB cell lines. A co-hu-HSAN-PBD ADC showed potent and specific cytotoxicity across GFRA2+ cell lines (NB=2; median IC50=1.69;2.92pM, EWS=2; median IC50=9.81;12.43pM) with no cytotoxicity in GFRA2 null cell lines (N=2; median IC50=NA). Conclusions: GFRA2 is a lineage restricted oncoprotein abundantly expressed in both NB/EWS, as well as other human solid cancers. We show initial proof-of-concept for potent and specific cytotoxicity in both NB/EWS with the co-hu-HSAN-PBD ADC. We will report on efficacy testing of the co-hu-HSAN-PBD ADC across NB/EWS xenograft models. Work to affinity enhance the fully human binders, and progress with additional linker/payload combinations are ongoing and will be reported. Citation Format: Amber K. Hamilton, Seungmin Shin, Raphael D. Lopez, Nicholas Hartnett, Alexander B. Radaoui, Maggie Hines, Maria Evancho, Rebecca S. Kaufman, Khushbu Patel, Karina L. Conkrite, Dan Martinez, Brian Mooney, Michelle E. Keyel, Elissa Levine, Alberto D. Guerra, Jarrett Lindsay, Yael P. Mosse, Jennifer Pogoriler, Gregg Morin, Poul H. Sorensen, Patrick J. Grohar, Benjamin A. Garcia, C. Patrick Reynolds, Wei Li, Sharon J. Diskin, John M. Maris. Development of a GFRA2-targeting antibody drug conjugate for neuroblastoma (NB), and Ewing sarcoma (EWS) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7805.

Although many DNA binding natural products exert their effects through non-specific mechanisms, a therapeutic opportunity exists for a subset of these compounds that alter the expression or activity of specific driver oncogenes in specific cell contexts. In this study, we integrate CUT&Tag with Global Run-On Sequencing (CUT, Tag, and GRO) to show that the minor groove binding compound, mithramycin (MMA), inhibits the Ewing sarcoma oncogenic driver, the EWS::FLI1 transcription factor. MMA causes either an increase or decrease in EWS::FLI1 binding to chromatin at downstream target response elements to poison nascent transcription. The reversal of EWS::FLI1 activity is limited by non-specific effects of the drug on RNAPII processivity but can be optimized by continuous administration at low concentration to cause more precise reversal of the oncogenic transcriptome and striking Ewing sarcoma xenograft regressions. The activity in vivo is further improved with a less-toxic second-generation analog, AIT-102.

Despite well-recognized biological heterogeneity, osteosarcoma has been treated as a single disease for over four decades with minimal improvement in survival. Clinical features are inadequate for risk stratification, and no molecular classifiers guide therapy. An international working group evaluated candidate prognostic biomarkers for clinical translation. Pre-treatment circulating tumor DNA is positioned for clinical implementation, while additional classifiers warrant prospective validation. This work establishes a path to risk-adapted, biologically informed treatment.

Ewing sarcoma (ES) is a bone and soft tissue sarcoma that is absolutely dependent on the EWS::FLI1 transcription factor for cell survival. No compound has been shown to reverse EWS::FLI1 activity in patients, and outcomes for relapsed patients remain poor. Trabectedin above a threshold concentration reverses the activity of EWS::FLI1 and is potentiated by low-dose irinotecan in vivo. This open-label phase 1/2 trial of trabectedin with irinotecan (SARC037) enrolled 37 relapsed/refractory patients with ES. The primary objectives were to determine the safety, tolerability, recommended phase 2 dose (RP2D; phase 1) and objective response rate (ORR; phase 2) of trabectedin administered as a 1-hour infusion in combination with low-dose irinotecan in patients with ES. The secondary objectives were to determine the progression-free survival (PFS), 6-month PFS, duration of response and 18F-fluorothymidine positron emission tomography (18F-FLT PET) avidity of ES tumors. The RP2D was trabectedin 1.0 mg m-2 over 1 hour (day 1) and irinotecan 25 mg m-2 (days 2 and 4) of a 21-day cycle. Toxicities were manageable with grade 3 or higher toxicities (>15%) of myelosuppression and alanine aminotransferase elevations at RP2D. The phase 2 ORR was 33% (39%, including RP2D phase 1 patients), and 6-month PFS was 48%. Transcriptional profiling demonstrated reversal of the EWS::FLI1 transcriptome in tumors from a subset of patients. Additional correlative objectives captured molecular profiling, circulating tumor DNA levels, pharmacokinetics and 18F-FLT PET avidity. Here we provide the basis for further development of trabectedin/irinotecan for patients with ES by the international cooperative groups. ClinicalTrials.gov: NCT04067115 .

Abstract Ewing sarcoma is an aggressive bone and soft-tissue cancer affecting adolescents and young adults. In vitro and in vivo models of Ewing sarcoma have been instrumental in advancing our understanding of Ewing sarcoma biology and essential in evaluating potential therapies, particularly for metastatic or relapsed disease for which effective treatment options remain limited. Through an international collaborative effort between the Children’s Oncology Group Bone Tumor Committee and the Euro Ewing Consortium, we review the current landscape of preclinical modeling used in Ewing sarcoma research encompassing both in vitro (cell lines and tumor organoids) and in vivo (mouse and nonmammalian xenografts) model systems. We discuss factors that can influence experimental results, provide testing considerations for both in vitro and in vivo studies, and descriptions of existing preclinical data repositories. We highlight current needs in Ewing sarcoma modeling and the importance of enhanced international cooperative research and patient advocacy efforts which will be critical in expanding our resources of biologically relevant Ewing sarcoma models to enable translation of preclinical findings into effective therapeutic strategies for patients with Ewing sarcoma.

Abstract Relapsed Ewing sarcoma has dismal outcomes and has had minimal treatment advancements in the last 30 years - there is a critical need for the development of new treatments. The tumor is uniquely dependent on the oncogenic EWS::FLI1 fusion transcription factor to develop and maintain malignancy. Lurbinectedin is a small molecule analog of the natural product trabectedin that has shown efficacy in multiple cancers including Ewing sarcoma. The mechanism of toxicity of lurbinectedin in Ewing sarcoma is under investigation. Lurbinectedin exposure alters EWS::FLI1 trafficking into the nucleolus as measured by confocal microscopy. The subsequent impact of drug exposure on EWS::FLI1 binding to chromatin was determined by CUT&Tag and confirmed by chromatin fractionation. Transcription was assessed by qPCR and RNAseq, and BRUseq was utilized to determine the impact on nascent transcription. Further mechanistic clarification was determined by proximity ligation mass spectrometry. Ultimately, the integration of CUT&Tag of EWS::FLI1 with Bru-seq (CUT, Tag, and Bru) following treatment with lurbinectedin elucidated the effects of the drug on EWS::FLI1 binding and alteration of downstream transcription. Exposure of Ewing sarcoma cells to lurbinectedin alters EWS::FLI1 distribution in the nucleus to the nucleolus. The relocalized EWS::FLI1 can be trapped in the nucleolus by use of potentiators that inhibit HSP70. The relocalization is driven by altered MAPK signaling and can be rescued by siRNA silencing of MAPK pathway members. The effect is rooted in wild-type function of the FET family of proteins and is associated with altered EWS::FLI1 binding at response elements leading to alteration of the transcriptome. Both transcription initiation and elongation are altered. Importantly, these effects on EWS::FLI1 are both dose and time dependent and can be further amplified with MAPK perturbants. Treatment with lurbinectedin induces relocalization of the oncogenic transcription factor EWS::FLI1 to the nucleolus leading to transcriptional suppression of EWS::FLI1 target genes and a striking decrease in cell viability. This modulation of EWS::FLI1 binding to chromatin occurs in both a concentration and time dependent manner. Intriguingly, the impact appears to be dependent on gene length: there is greater transcription alteration in longer genes compared to shorter genes. Citation Format: Zachary P. Tolstyka, Raphael D. Lopez, Sridhar M. Veluvolu, Emma Hiscock, Andrew Fuller, Mia Lollo, Emily Seiden, Rachael Hinshaw, Lauren Gaetano, Elizabeth Wilson, Gretchen Lam, Rebecca Kaufman, Elissa Boguslawski, Michelle Paulsen, Ishwarya Narayanan, Jenna Gedminas, Mats Ljungman, Patrick J. Grohar. Lurbinectedin alters EWS::FLI1 binding to chromatin to poison transcription [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4776.

PURPOSE:Identifying discrete subgroups associated with treatment response and resistance in localized Ewing sarcoma (EWS) remains a challenge. The primary objective of the Children's Oncology Group (COG) biology study AEWS18B1-Q was to molecularly characterize patients with localized EWS on prospective modern-day trials. PATIENTS AND METHODS:We analyzed clinical and molecular features from patients with localized EWS enrolled on frontline COG trials. All patients had available formalin-fixed paraffin-embedded (FFPE) tissue, frozen tissue, or whole-genome-amplified material. Sequencing was performed for identification of canonical fusions, recurrent copy number alterations (CNAs), and alterations in TP53 and STAG2. Available tissue was analyzed for loss of STAG2 protein expression. Molecular features were evaluated for their association with cumulative incidence of relapse in univariate and multivariable analyses. RESULTS:Three hundred fifty-one patients had sufficient tissue, which in most cases was extracted from two FFPE slides. EWS canonical fusions were identified in 282 patients (80.3%). Pathogenic mutations in TP53 and STAG2 were identified in 5.1% and 7.6% of patients, respectively. A total of 63.1% of patients were found to have recurrent CNAs. In univariate analysis, there was an increased cumulative incidence of relapse in patients with TP53 mutation (5-year cumulative incidence of relapse 43%, 95% CI [17% to 67%] v 22%, 95% CI [17% to 27%]; Gray's test P = .039), STAG2 mutation (53%, 95% CI [29% to 73%] v 21%, 95% CI [16% to 26%]; P < .001), and recurrent CNAs (30%, 95% CI [22% to 37%] v 16%, 95% CI [9% to 24%]; P = .005). In a multivariable analysis, STAG2 mutation was the only molecular biomarker that remained prognostic. CONCLUSION:This is a prospective validation of the molecular prognostic features of patients with localized EWS receiving standard-of-care therapy on therapeutic clinical trials. Building on previous work, patients with STAG2 mutations were at high risk of relapse.

Ewing sarcoma is an aggressive bone and soft-tissue cancer affecting adolescents and young adults. In vitro and in vivo models of Ewing sarcoma have been instrumental in advancing our understanding of Ewing sarcoma biology and essential in evaluating potential therapies, particularly for metastatic or relapsed disease for which effective treatment options remain limited. Through an international collaborative effort between the Children's Oncology Group Bone Tumor Committee and the Euro Ewing Consortium, we review the current landscape of preclinical modeling used in Ewing sarcoma research encompassing both in vitro (cell lines and tumor organoids) and in vivo (mouse and nonmammalian xenografts) model systems. We discuss factors that can influence experimental results, provide testing considerations for both in vitro and in vivo studies, and descriptions of existing preclinical data repositories. We highlight current needs in Ewing sarcoma modeling and the importance of enhanced international cooperative research and patient advocacy efforts which will be critical in expanding our resources of biologically relevant Ewing sarcoma models to enable translation of preclinical findings into effective therapeutic strategies for patients with Ewing sarcoma.

Outcomes for relapsed Ewing sarcoma remain consistently poor. Continued efforts to consider creative new approaches for the treatment of relapsed Ewing sarcoma are needed. The Children's Oncology Group Bone Tumor Committee convened a New Strategies for Ewing Sarcoma Task Force to systematically evaluate agents for inclusion in future Phase II or III clinical trials for relapsed Ewing sarcoma. In addition, the Task Force summarized the possible limitations of common trial designs and considered new approaches to study promising agents in clinical trials. Here, we summarize this work and propose next steps for future trials for relapsed Ewing sarcoma.

Abstract Background: Ewing sarcoma (ES) is a malignant bone tumor characterized by the oncogenic fusion protein EWS-FLI1. EWS-FLI1 acts as an aberrant transcription factor, inducing and silencing genes involved with increasing cell proliferation and survival. We have previously shown that lurbinectedin, a trabectedin analog, effectively inactivates the transcriptional activity of EWS-FLI1 by redistributing it within the nucleus to the nucleolus. Interestingly, this process activates a gene signature like that of the DNA damage response (DDR) to UV light, which was also shown to induce nucleolar translocation of WT Ewing sarcoma breakpoint 1 (EWSR1). However, the mechanism governing the nucleolar translocation of EWS-FLI1 induced by these drugs has yet to be elucidated. In addition, novel approaches to exploit this relocalization mechanism as a means to broaden the therapeutic window have yet to be established. Methods: We used immunoblotting, RT-qPCR, viability assays, cell fractionation, confocal microscopy, proximity ligation assays (PLA), mass spectrometry, genomics, and immunoprecipitation to investigate the mechanism of action of trabectedin/lurbinectedin-induced nucleolar relocalization of EWS-FLI1. Results: Through interrogation of the pathways triggered by trabectedin/lurbinectedin, we found that this mechanism is in part mediated by HSP70. Viability assays also revealed profound synergy between HSP70 inhibitors (HSP70is) and lurbinectedin. Immunoprecipitated EWS-FLI1 subjected to mass spectrometry revealed an association of EWS-FLI1 and several HSP70 isoforms after trabectedin treatment which was confirmed with PLA. Trabectedin/lurbinectedin treatment also revealed an upregulation of EWS-FLI1-suppressed targets and suppression of induced targets. Confocal microscopy showed that EWS-FLI1 “trapping” in the nucleolus was potentiated by combination treatment with HSP70is. Conclusion: Our results underscore the synergistic effect of combining HSP70is with trabectedin or lurbinectedin treatment to attenuate several aspects of ES tumorigenesis in vitro. In vivo experiments with patient-derived xenografts are underway to corroborate the synergy of HSP70is and trabectedin/lurbinectedin. Together, our data highlights the potential for a novel “EWS-FLI1 nucleolar trap” as a means to inhibit and sustain EWS-FLI1 repression with this class of compounds. Citation Format: Sridhar M. Veluvolu, Raphael D. Lopez, Jenna M. Gedminas, Elissa A. Boguslawski, Elizabeth R. Wilson, Benjamin P. Caiello, Maureen E. Murphy, Patrick J. Grohar. Exploiting lurbinectedin-driven nucleolar relocalization of EWS-FLI1 to develop novel combination therapies for 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 1082.

Trends in diagnostic biopsy sample collection approaches for primary bone sarcomas have shifted in the past 2 decades. Although open/incisional biopsies used to be the predominant approach to obtain diagnostic material for Ewing sarcoma and osteosarcoma, image-guided core needle biopsies have increased in frequency and are safe for patients. These procedures are less invasive and reduce recovery times but have potential limitations. The quantity and quality of tissue obtained through these procedures vary between institutions. Acquired viable tissue volumes can be low, limiting the conduct of downstream expanded clinical workup, molecular analyses, and research. Patients with advanced Ewing sarcoma and osteosarcoma continue to have overall poor outcomes despite dose-intensive cytotoxic chemotherapy. The biology of treatment resistance is not currently well understood, partly due to limited availability of relevant tissue to study. There is a need for access to quality tumor specimens for molecular and other analyses to identify high-risk tumor subsets and drive discovery to improve patient outcomes. Given broad variability in bone tumor tissue procurement and processing across member institutions, the Children’s Oncology Group Bone Tumor Committee convened a multidisciplinary group of experts to outline the current and near-future tissue needs for optimal clinical care and access to research platforms. The goal of this working group was to provide high-level guidance on biopsy practices that safely meet these evolving needs. Harmonizing tissue collection practices is paramount to improving the care of children, adolescents, and young adults diagnosed with Ewing sarcoma and osteosarcoma.

Desmoplastic small round cell tumor (DSRCT) is a highly aggressive cancer predominantly occurring in male adolescents and young adults. The lack of a comprehensive understanding on the biology of the disease is paralleled by its dismal survival rates (5–20%). To overcome this challenge, we first identified and prioritized urgently needed resources for clinicians and researchers. Thus, we established genome-wide single-cell RNA-sequencing and bulk proteomic data of in vitro and in vivo-generated knockdown models of the pathognomonic DSRCT fusion oncoprotein (EWSR1::WT1) and combined them with an original systems-biology-based pipeline including patient data and the largest histology collection of DSRCTs and morphological mimics available to date. These novel tools were enriched with curated public datasets including patient- and cell line-derived ChIP-seq, bulk and single-cell RNA-seq studies resulting in a multi-model and multi-omic toolbox for discovery analyses. As a proof of concept, our approach revealed the alpha-2/delta subunit of the voltage-dependent calcium channel complex, CACNA2D2, as a highly overexpressed, super-enhancer driven, direct target of EWSR1::WT1. Single-cell and bulk-level analyses of patient samples and xenografted cell lines highlighted CACNA2D2 as a critical component of our newly established EWSR1::WT1 oncogenic signature, that can be employed to robustly identify DSRCT in reference sets. Finally, we show that CACNA2D2 is a highly sensitive and specific single biomarker for fast, simple, and cost-efficient diagnosis of DSRCT. Collectively, we establish a large-scale multi-omics dataset for this devastating disease and provide a blueprint of how such toolbox can be used to identify new and clinically relevant diagnostic markers, which may significantly reduce misdiagnoses, and thus improve patient care. ### Competing Interest Statement The authors have declared no competing interest.

Abstract Background: Tumor necrosis factor receptor superfamily member 10b (TNFRSF10b, also known as DR5) agonists are attractive targeted agents for cancer cells because these agents activate cellular apoptosis. Ewing sarcoma (ES) is a bone and soft-tissue sarcoma occurring in adolescence defined by a t(11;22)(q24;q12) balanced chromosomal translocation that results in chimeric transcription factor and oncogenic driver protein, EWS-FLI1. Independent studies and unbiased cell line screens have reported ES to be sensitive to DR5 targeting. INBRX-109 is a tetravalent, DR5-targeted antibody. Here, we analyzed the sensitivity of ES models to DR5 targeting by INBRX-109 and explored how the EWS-FLI1 transcriptional program impacts the continuum of sensitivity-resistance to this targeting. Methods: In a panel of 11 ES lines, in vitro sensitivity was assessed to both single agent and INBRX-109 combinations. A screen of 1,363 FDA approved compounds identified cooperative combinations that were prioritized mechanistically. Differential sensitivity for single agent and in combination with irinotecan was confirmed in vivo in xenograft models. Resistant clones were isolated from in vivo experiments and evaluated for DR5 expression and sensitivity. Cellular factors associated with sensitivity and resistance were determined by siRNA knockdown and Cleavage Under Targets and Tagmentation assay (CUT and Tag). Results: ES models showed variable sensitivity to INBRX-109, with a GI90 as low as 10 picomolar in some models. All models showed robust expression of DR5, but absolute expression of DR5 did not correlate with sensitivity. In vitro sensitivity was replicated in vivo, with all 7 mice bearing RD-ES xenografts demonstrating initial complete tumor regression and 3 of 7 mice demonstrating no recurrence beyond 90 days. Importantly, cells isolated from recurrent tumors showed a novel mechanism of resistance: direct repression of DR5 by EWS-FLI1. INBRX-109 was ineffective as a single agent at 10 nM in resistant ES models. However, there was striking synergy in vitro with SN38, the active metabolite of irinotecan, and this effect translated into tumor regressions of a resistant TC32 xenograft in vivo. The FDA compound screen captured novel synergy with a panel of agents. Conclusion: INBRX-109 is an exciting candidate for ES as a single agent in subsets of tumors or in combination with irinotecan, which is currently being evaluated in the clinic. Investigation of the exact relationship between EWS-FLI1 and sensitivity to DR5 targeting is ongoing. Initial findings suggest EWS-FLI1, combined with other disease modifying mutations, is a key mediator of this sensitivity. Further, EWS-FLI1 plays a direct role repressing DR5 in the setting of therapeutic challenge in one model in vivo. These findings highlight the potential for INBRX-109 in combination with irinotecan or novel candidates as therapies for ES patients. Citation Format: Curtis C. Parker, Lauren M. Gaetano, Elissa A. Boguslawski, Seneca Kinn-Gurzo, Rebecca Kaufman, Matthew C. Stout, Chase Deveraux, Patrick J. Grohar. A role for the oncogenic driver fusion protein EWS-FLI1 in the targeting of DR5 by INBRX-109 in 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 1081.

Supplementary Figures S1-S15 and Legends