Ewing Sarcoma Research Articles

Abstract 2862: Evolution of clinically relevant variants in advanced pediatric sarcomas

Abstract Objective Few pediatric sarcoma studies have investigated how gene alterations detected by targeted sequencing change in the same patient over time. We leveraged targeted somatic DNA next-generation sequencing (NGS) data from a large cohort of young patients with osteosarcoma (OS), Ewing sarcoma (EWS), or rhabdomyosarcoma (RMS) to identify changes in known oncogenic variants with the aim of addressing the clinical utility of resequencing at recurrence. Methods The iCat2/GAIN Consortium Study is a cohort study involving 13 US institutions evaluating outcomes after receipt of matched targeted therapy (MTT) in young patients with advanced extra-cranial solid tumors. Tumors are sequenced using OncoPanel, a targeted DNA NGS test performed in a CLIA/CAP certified laboratory assessing single nucleotide variants (SNV), copy number variants (CNV) and structural variants (SV) in up to 447 cancer genes and rearrangements in up to 60 cancer genes. In 2 cases, similar targeted NGS tests were performed. For OS, EWS, or RMS patients with at least 2 OncoPanels from different timepoints (initial diagnosis or relapse) and/or tumor sites (metastatic or primary), we assessed oncogenic variants in genes previously known to be recurrently altered in the respective diagnosis. For each variant, we assigned patterns of change as emergence, disappearance, or persistence. Results Of 784 iCat2/GAIN patients, there were 157, 107, and 100 patients with OS, EWS, and RMS respectively. The analytic cohort consisted of 34, 20, and 15 OS, EWS, and RMS patients respectively with at least 2 tumor sequenced samples. The median number of sequenced samples per patient was 2 (range 2 to 6). In EWS, 9 (45%) patients had an oncogenic variant (other than an expected fusion) in at least one sample. In 4 patients, the predominant pattern of change was emergence. Specifically, a new pathogenic SNV in STAG2 (N=1) or TP53 (N=3) were observed. In OS, all 34 patients had an oncogenic variant in at least one sample. In RMS, 12 (80%) patients had an oncogenic variant in at least one sample. There was no predominant pattern of change in OS and RMS. In OS, 41% percent of variants persisted in all samples; 32% of variants disappeared in subsequent samples; 22% of variants emerged in subsequent samples; 5% of variants were variably present over time. Interestingly, while TP53 was most often persistent over time, there were also 7 cases with emergence of TP53 variants and 8 cases with disappearance of TP53 variants. In RMS, 26% percent of variants persisted in all samples; 31% of variants disappeared in subsequent samples; 43% of variants emerged in subsequent samples. Interestingly, in three cases there was emergence of a new variant activating the RAF/MAPK or AKT pathway. Conclusion Pediatric sarcomas display a range of genomic changes over time. Given the observed changes in oncogenic alterations, there may be utility to repeat sequencing of relapsed tumor biopsies to understand these changes. Citation Format: Hannah Y. Comeau, Lorena Lazo De La Vega, Alanna J. Church, Luke D. Maese, Navin Pinto, Margaret E. Macy, Mark A. Applebaum, Amit J. Sabnis, AeRang Kim, Susan I. Colace, Filemon Dela Cruz, Steven G. DuBois, Natalie B. Collins, Katherine A. Janeway. Evolution of clinically relevant variants in advanced pediatric 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 2862.

Abstract 1560: In vivo CRISPR/Cas9 transcriptional activation screen leads to discovery of INAFM2, a novel driver of metastasis

Abstract Metastasis is the main cause of mortality in cancer patients and accounts for about 90% of cancer deaths, which has not changed in the past 50 years. This indicates a significant need for further study of metastasis pathways to find novel druggable targets. Ewing sarcoma (ES) is an aggressive childhood cancer that occurs in the bones or soft tissue. The five-year survival rate for Ewing sarcoma patients diagnosed with metastatic disease is still only 25%, making ES a good model for studying metastasis. We performed a genome-wide CRISPR/Cas9 transcriptional activation screen in a zebrafish xenograft model to discover genes whose activation leads to increased metastasis. The screen identified a number of genes that may accelerate ES metastasis; we chose INAFM2 for further study because multiple unique INAFM2-targeting gRNAs were enriched in the screen and we discovered a potential link to metastasis and patient survival in publicly available clinical data. Overexpression of INAFM2 in six different cancer cell lines (four ES, one hepatocellular carcinoma, and one stomach adenocarcinoma) caused increased cell migration and chemotaxis in vitro, and increased intravasation in zebrafish xenografts. INAFM2 knockdown in four cancer cell lines (two ES, one clear cell renal cell carcinoma, and one stomach adenocarcinoma) decreased chemotaxis and rescue of INAFM2 expression restored the cells’ ability to migrate. Analysis of patient tumor RNA sequencing data from many different types of cancers revealed correlations between INAFM2 expression and both an increased metastatic phenotype and poor patient survival. Further in vivo studies by tail vein injection of ES cells in immunodeficient mice demonstrated significantly increased gross metastases with INAFM2 overexpression and decreased gross metastases with INAFM2 knockdown compared to the respective control groups. This provides strong evidence for a substantial role of INAFM2 in cancer metastasis. Human INAFM2 is an unstudied gene and our preliminary work has revealed that the INAFM2 protein localizes to the cell membrane, is glycosylated, and may be involved in Wnt and/or MAPK pathway signaling. Inafm2 silencing by morpholino in zebrafish embryos lead to significant mortality, necrosis, and developmental delays/defects including severe cardiac edema. Current studies are focused on further discerning the molecular pathways that mediate the role of INAFM2 in promoting metastasis using in vitro and in vivo models as well as characterizing the functional significance of the direct protein binding partners and post-translational modifications for INAFM2. Citation Format: Anna Molotkova, Emre Deniz, Matthew Swift, Eric Glasgow, Jeffrey Toretsky, Aykut Üren. In vivo CRISPR/Cas9 transcriptional activation screen leads to discovery of INAFM2, a novel driver of metastasis [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 1560.

Abstract 1081: A role for the oncogenic driver fusion protein EWS-FLI1 in the targeting of DR5 by INBRX-109 in Ewing sarcoma

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.

Abstract 1787: The β3-adrenergic receptor as novel target of metabolic phenotype in Ewing sarcoma

Abstract Purpose of study: Metabolic targeted therapies may represent an innovative strategy in Ewing sarcoma (ES). Introduction: Ewing sarcoma (ES) is highly aggressive round cell mesenchymal neoplasm, most often occurring in children and young adults. Metabolic peculiarities of ES cells are indeed involved in tumor growth, survival and resistance to therapy. We have further investigated apoptotic role of 𝜷;;3-adrenergic receptor (𝜷;;3-AR) SR59230A (SR) antagonist for its ability to reduce cells viability due increased oxidative stress, now we underline the metabolic role of SR. Materials & Methods: The metabolic profile after SR administration in A673 ES cell model was studied using the Seahorse XF Analyzer. Viability was evaluated with MTS. Changes in gene expression was evaluated with RNA sequencing. Western blot analysis and Immunofluorescence was performed for redox signature and metabolic alteration. Ros production and cell death was assessed by flow cytometry. Results: Metabolic analysis reveals glucose and fatty acid dependency in A673 cells using the Fuel Flex Test of Seahorse XFe Analyzer. Quantification of transcript abundance using Salmon in A673 after SR treatment showed decrease in response to reactive oxygen species, increase of Thioredoxin Interacting Protein (TXNIP) inhibits the antioxidative function of thioredoxin, moreover reduced glucose uptake, consequently, decrease NADPH/NADP resulting in the accumulation of ROS coincident with a decrease of GLUT1, SOD2, TrDX an increase of LC3IIA protein expression. SR enhances A673 pyruvate dependency, and decrease glyco and mito ATP production rate, more over observed under glucose deprivation. Furthermore, apoptotic effect was significantly amplified when cells were under nutrient starvation, revealing a 56% apoptotic rate after 24 hours of SR treatment. Conclusion: SR antagonist of 𝜷;;3-AR and combination of SR with BUF could be an innovative therapeutic option to raise awareness ES. Citation Format: Cristina Banella, Francesco Carrozzo, Laura Zocca, Amada Pasha, Gianluca Mattei, Marina Mola, Lara Ballerini, Nicla Lorito, Annalisa Tondo, Claudio Favre, Maura Calvani. The β3-adrenergic receptor as novel target of metabolic phenotype 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 1787.

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.

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 5777: Rewiring EWS/FLI1 with transcriptional chemical inducers of proximity (TCIPs)

Abstract The purpose of this study was to determine if EWS/FLI1 (E/F), the oncogenic fusion protein that drives Ewing Sarcoma (ES), can be rewired to enhance expression of BCL6-controlled genes using Transcriptional Chemical Inducers of Proximity (TCIPs), a novel bivalent small molecule platform. TCIP molecules recruit a transcriptional regulator, such as BRD4, to the transcriptional repressor BCL6. Upon binding, a ternary complex is formed on chromatin at BCL6-bound loci, causing transcriptional upregulation of BCL6 target genes. Since E/F does not have a bona fide ligand, we have used an exogenously expressed N terminal FKBPF36V-EWS/FLI1 (N-FK-E/F) fusion system to study how modulation of E/F by TCIPs impacts the transcriptome of ES cells. To eliminate background signal from endogenous E/F, we used CRISPR/Cas9 to knockout (KO) endogenous E/F. Our exogenously expressed N-FK-E/F has mutated PAM motifs and is resistant to KO. In our proof-of-concept study we used TCIP1 consisting of ortho-AP1867 (oAP), a synthetic ligand for FKBPF36V, linked to the BCL6 inhibitor BI3812 (BI) to rewire N-FK-E/F. We have also tested a negative control TCIP (TCIP NEG) that links oAP to a BI derivative that abolishes BCL6 binding. TCIP NEG has similar physiochemical properties to our active TCIP1; however, it cannot bind BCL6, serving as a negative control for ternary complex formation. TCIP1 rapidly and dose-dependently increases the RNA and protein expression of several BCL6 target genes, like p21, in multiple ES models that stably express N-FK-E/F. TCIP NEG does not increase the expression of BCL6 target genes in ES cells. TCIP1 induces ternary complex formation in ES cell lysates, and ternary complex formation is necessary for activity in ES cells. TCIP1 is specific and does not increase BCL6 target gene levels to the same extent in ES cells expressing a FKBP-GFP construct or in parental ES cells lacking N-FK-E/F. RNA sequencing of EWS502 N-FK-E/F expressing cells shows that TCIP1 treatment induces a larger increase in expression of BCL6 target gene RNA than BCL6 inhibition. Moreover, CUT&RUN experiments in EWS502 N-FK-E/F expressing cells suggest TCIP1 leads to an accumulation of N-FK-E/F at the promoters of BCL6 target genes. Motif analysis showed that TCIP1 increases the likelihood of N-FK-E/F binding to BCL6 consensus sequences as wells as increases the likelihood of BCL6 binding to E/F recognition sequences. In future studies we will conduct RNA sequencing, CUT&RUN, and other ‘omics’ approaches (e.g. DisP-seq) to compare TCIP1 to TCIP NEG in multiple cell lines at multiple concentrations and time points. Our data suggests that N-FK-E/F can be rewired to induce the expression of BCL6 target genes in ES. Since ES is driven solely by E/F, TCIPs may be promising next generation therapeutics. Our study provides a proof-of-concept that will be the foundation for the development of future TCIP molecules that recruit endogenous E/F once suitable ligands are developed. Citation Format: Michael J. Bond, Ryan P. Golden, Roman C. Sarott, Giulia DiGiovanni, Basel A. Karim, Briana Howard, Kenneth Ross, Nathanael S. Gray, Kimberly Stegmaier. Rewiring EWS/FLI1 with transcriptional chemical inducers of proximity (TCIPs) [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 5777.

Abstract 4607: Targeted nanoparticle delivery of irinotecan and Ewing sarcoma-specific siRNA is an effective combination therapy for Ewing sarcoma

Abstract Background: Ewing sarcoma (ES) is an aggressive bone and soft tissue tumor affecting children and adolescents with an overall survival rate of 50%. Irinotecan (IR) is an effective chemotherapeutic agent but is associated with significant toxicity. Using an anti-CD99 targeted hybrid polymerized lipid nanoparticle containing IR, NV103, in pre-clinical mouse xenograft model of the disease, tumor ablation was achieved at 2mg/kg of IR versus 50mg/kg for free IR. All ES harbor a chromosomal translocation resulting in expression of a fusion EWS-FLI1 protein or equivalent. This chimeric gene is a transcriptional regulator that upregulates the FEZF1 and FEZF1-AS1 genes. Both EWS-FLI1 and FEZF1-AS1 genes are unique to ES and are not expressed in normal adult tissues. EWS-FLI1 promotes cell growth while the FEZF1-AS1 complex promotes tumor metastasis, a hallmark of ES. In vitro RNAi targeting of EF and FEZF1-AS1 results in tumor cell death. We hypothesized that combining EWS-FLI1 and/or FEZF1-AS1 targeted RNAi therapy with NV103 cytotoxic therapy would be more effective than any single modality therapy. Methods: NV103 was provided by NanoValent Pharmaceuticals. siRNA containing lipid nanoparticles (LNP-siRNA) were generated using PreciGenome NanoGenerator. siRNA was quantified using the ribogreen assay. Nanoparticles were analyzed for size and particle numbers by NanoSight. siRNA was used to target the following genes- EWS-FLI1, FEZF1, FEZF1-AS1, or GFP. A673 cells expressing GFP were used for this in vitro study. Various doses of IR and siRNA were used in combination and cell growth/death was imaged live using Incucyte. ATP assay was used to confirm live cells and to determine minimum effective dose (MED) over 72 hours of treatment. Results: LNP-siRNA, mean diameter 70nm and PDI<0.2, encapsulated 90% of the siRNA in solution. LNP are stable at 4°C for at least 4 weeks with minimal-to-no loss of activity. NV103 had a MED of 1µM. MED of various siRNA was 30 to 50nM. Combination of NV103 and LNP-siRNA reduced MED of IR to 250nM. One treatment dose of LNP-siRNA followed by NV103 treatment three days later maintained significant cell death over 10 days post treatment initiation. Control cells were 97% more confluent than NV103/EWS-FLI1 siRNA treated ones, 85% more than NV103/FEZF1 siRNA cells and 92% more than NV103/FEZF1-AS1 treated cells. siRNA combinations of EWS-FLI1 and FEZF1 or FEZF1-AS1 along with NV103 added no further benefit. Conclusion: Combination nanoparticle therapy of IR and ES-specific siRNA reduces IR MED by four-fold. LNP delivery of siRNA enables tumor-specific gene targeting. Most have no available small molecule therapy. Targeting ES cells with both a low dose (250nM) of IR and 30-50nM of siRNA against unique tumor biomarkers results in increased cell death with no IR toxicity. Further animal studies are required to determine the effectiveness of such therapy in vivo. Citation Format: Sheetal A. Mitra, Jean-Hugues Parmentier, Hyung-Gyoo Kang, Timothy J. Triche. Targeted nanoparticle delivery of irinotecan and Ewing sarcoma-specific siRNA is an effective combination therapy 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 4607.

Abstract 5458: Plasticity of CAF-like states in Ewing sarcoma is mediated by TGF-beta

Abstract Transcriptional plasticity of cancer cells promotes tumor progression and treatment resistance via generation of phenotypically distinct cell states. We previously showed that Ewing sarcoma (EwS) cells exhibit inter- and intra-tumoral transcriptional heterogeneity and that they undergo state changes in response to cues from the tumor microenvironment (TME). More recently, we identified phenotypically distinct EwS tumor cell subpopulations in vivo that deposit pro-tumorigenic extracellular matrix (ECM) proteins, adopting similar functions to cancer-associated fibroblasts (CAFs). Here, we sought to identify the upstream regulators of these CAF-like EwS cells with the goal of defining molecular mechanisms that control EwS plasticity and creation of ECM-remodeling states. Integration of single cell proteogenomic sequencing (CITE-seq) with immunofluorescence and digital spatial profiling of cell line xenografts and patient tumors validated the cell-surface protein CD73 (NT5E) as a biomarker of ECM-secreting EwS cell subpopulations in vitro and in vivo. The transcriptomes of CD73+ CAF-like cells were compared to isogenic CD73- cells (n=9 cell lines) and highly significant enrichment of gene ontology terms related to TGF-beta signaling (p.adj = <4.4x10-14) was noted. EwS cells were previously reported to be non-responsive to TGF-beta due to profound transcriptional repression of the TGF-beta type 2 receptor (TGFBR2) by EWS::FLI1. In support of this, TGFBR2 expression was low in bulk measurements of EwS cells. However, TGFBR2 mRNA and surface protein expression were significantly increased in CD73+ subpopulations. Exposure of EwS cells to TGF-beta ligands (TGFB1 or TGFB2) strongly induced the CAF-like gene signature, including ECM genes BGN, COL1A1, SPARC, and TNC (p. adj < 8.5x10-5), and this was augmented in CD73+ cell populations. Transduction of EwS cells with dominant negative-TGFBR2 inhibited both basal and TGF-beta-induced expression of CAF-like signature genes and blocked SMAD2 phosphorylation. To test the functional significance of the TGF-beta response, unsorted EwS cells were plated in 2D or as tumor spheroids in collagen I-rich 3D cultures and exposed to TGFB1 or SB-505124, a TGF-beta inhibitor. TGFB1 treatment had no effect on cell viability or proliferation in 2D but increased invasion into collagen in 3D. Inhibition of TGF-beta reduced CAF-like gene expression in 2D cultures and diminished both viability and invasion in 3D. Finally, our studies revealed that NT5E (CD73) is a TGF-beta induced target in EwS cells and that CD73+ cells upregulate expression of TGFB2. Thus, an autocrine signaling circuit is created in which TGF-beta activity in CAF-like tumor cell subpopulations is sustained by CD73+ tumor cell-derived ligand. Together these studies demonstrate that TGF-beta is a key determinant of cell plasticity and ECM remodeling in EwS. Citation Format: Emma Wrenn, April A. Apfelbaum, Katherine Braun, Erin R. Rudzinski, Xuemei Deng, Aya Miyaki, Trisha Lipson, Nicolas M. Garcia, Virginia J. Hoglund, Elizabeth R. Lawlor. Plasticity of CAF-like states in Ewing sarcoma is mediated by TGF-beta [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 5458.

Abstract 3244: EWS-FLI1-driven upregulation of UGT3A2 unlocks drug sensitivity pathway in Ewing sarcoma

Abstract Ewing sarcoma (ES), an aggressive pediatric bone tumor, presents a clinical challenge, particularly for metastatic or relapsed disease. The hallmark genetic alteration involves the fusion of the FET RNA-binding protein, EWSR1, with an ETS transcription factor, typically FLI1, resulting in the EWS-FLI1 fusion oncoprotein. EWS-FLI1 orchestrates a unique epigenetic program, leading to the aberrant expression of genes crucial for cell-cycle regulation, DNA damage response, and cell migration. Targeting the EWS-FLI1 fusion oncoprotein is a daunting task, which has prompted us to pursue other strategies in parallel. Here, we present an innovative approach harnessing the power of phenotypic drug screens to uncover amenable pathways for targeted medicines in ES. Leveraging data from the Broad Institute’s repurposing dataset encompassing 4,518 drug molecules screened across 578 adherent cancer cell lines using the high-throughput multiplexed screening platform known as PRISM, we identified compounds with selective efficacy against ES. The top-ranking compound identified for selective ES toxicity in the screen was R112, an ATP-competitive inhibitor of Spleen Tyrosine Kinase (SYK). In low-throughput evaluation, R112 induced selective ES cell death when compared to other sarcomas. However, the reported target of R112, SYK, is a dependency in ES based on the Cancer Dependency Map (DepMap) resource. Subsequent validation of compounds identified in the PRISM screen as selectively toxic in ES revealed that while majority of them induced selective ES cell death, their reported targets were diverse and not linked to known dependencies in ES. Notably, one of the emerging compounds, luteolin, a natural compound known for its antioxidative properties, stood out and was selected for further studies alongside R112. Comprehensive genome-wide CRISPR-rescue screens identified UGT3A2, encoding a UDP glycosyltransferase, along with its solute carrier, SLC35B4, as pivotal genes that when deleted rescued both R112 and luteolin-induced cytotoxicity in ES cells. UGT3A2, a direct target gene of EWS-FLI1, demonstrated hyperexpression in EWS-FLI1-positive ES cells and no expression in most cancer and normal cells. While ES cells do not depend on UGT3A2 for survival, this abnormal elevation of UGT3A2 expression emerged as a key determinant driving their hypersensitivity to a subset of additional small molecules identified from the PRISM screen. Despite divergent mechanisms and direct targets, these drugs shared a common phenoxy motif, which UGT3A2 glycosylated. This specific EWS-FLI1-induced UGT3A2 upregulation, thus, unveils a novel biomarker for drug sensitivity,paving the way for precision medicine strategies tailored for Ewing sarcoma. Beyond Ewing sarcoma, this work holds promise in reshaping therapeutic paradigms and offering new avenues for discovering safe and enhanced treatments across a spectrum of cancers. Citation Format: Olasunkanmi Olaoye, Colleen Harrington, Kenneth Ross, Bo Kyung A. Seong, Luk de Waal, Jun Qi, Jennifer Roth, Kimberly Stegmaier. EWS-FLI1-driven upregulation of UGT3A2 unlocks drug sensitivity pathway 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 3244.

Abstract 4255: A systematic framework for efficient generation of expandable patient-derived 3D sarcoma models

Abstract Introduction: Sarcomas are rare connective tissue tumors with over 100 described subtypes. High heterogeneity within and among sarcoma sub-entities and low incidence contribute to poor therapeutic success. Patient-derived 3D models (PDMs), i.e. organoids and spheroids, closely recapitulate features of the original tumor, e.g. cell-to-cell interaction, differentiation potential and heterogeneity. However, PDMs are rarely available or even lacking for most sarcoma sub-entities, constituting a major hurdle for sarcoma precision oncology and research. Here, we present a highly efficient and systematic framework to generate long-term 3D models for distinct sarcoma subtypes, which we developed within the HEROES-AYA consortium. Methods: Tumors were received directly after surgery and processed within 24 hours. After enzymatic dissociation, cell clumps were seeded in 384-well plates with and without Matrigel. Each tumor was cultivated on an array of 140 distinct culture conditions, differing in contribution and concentration of pathway inhibitors and activators. Organoid and spheroid growth was assessed over time via imaging and AI-based algorithms to identify best-growing conditions. Results: Initially, we combined Ewing sarcoma (EwS) secreted factors and potentially relevant cytokines to generate a matrix of 140 diverse media, named Media Test Plate (MTP). The MTP was tested on five EwS tumors, allowing the identification of optimal culture conditions with 100% efficiency. When transplanted in NSG mice, the established PDMs showed tumorigenicity potential and Ewing sarcoma-specific CD99 marker expression. The MTP was then tested on four synovial sarcomas (SyS) and five myxoid liposarcomas with a 75% and 50% success rate, respectively. The optimal media, as identified by MTP, allowed the cultivation of one EwS and one SyS out of two, respectively, even with limited cell count. Additional PDMs generated include one undifferentiated, one alveolar soft tissue and one spindle cell sarcoma. Established cultures were characterized by Ki67 positivity, ranging from 40% to 70%, and expression of sarcoma subtype-specific markers. EwS models resulted 100% CD99 positive, while TLE1 positivity in SyS models was ≈90%. DNA and RNA sequencing of PDMs will show whether they resemble the genetic and transcriptional features of the original tumors. Conclusions: We here present a novel systematic protocol to generate organoids and spheroids from different sarcoma subtypes. To date, no such systematic protocol is available for sarcoma models generation. Our success rate is comparable to the one of intestinal and pancreatic organoids and exceeds those of 2D and PDX sarcoma cultures, which is ≈20%. Current work aims to adapt the MTP to a broad range of sarcoma subtypes, including rare entities. The efficient generation of expandable PDMs from a wide range of sarcomas will further fuel basic and translational research. Citation Format: Claudia Dagostino, Waqar Hussain, Jasmina Paluncic, Marcelo Zoccoler, Jessica Pablik, Daniela Richter, Lisanne Knol, Ana Banito, Johanna Wagner, Ivona Mateska, Daniel E. Stange, Ina Oehme, Stephan Richter, Stefan M. Pfister, Stefan Fröhling, Claudia Scholl, Jürgen Weitz, Klaus-Dieter Schaser, Christoph Heining, Hanno Glimm, Claudia R. Ball. A systematic framework for efficient generation of expandable patient-derived 3D sarcoma models [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 4255.

Abstract 901: BATCHIE: An active learning platform for scalable combination drug screens

Abstract Large-scale combination drug screens are largely considered intractable due to the immense number of possible combinations. Existing approaches use ad hoc fixed experimental designs then train machine learning models to impute novel combinations. We introduce BATCHIE, an orthogonal approach that adaptively conducts experiments in batches. BATCHIE uses information theory and probabilistic modeling to design each batch to be maximally informative based on the results of previous experiments. BATCHIE is fully modular, allowing any Bayesian probabilistic model to be used and any study constraints to be incorporated while maintaining optimality guarantees. Results: In retrospective simulations on public combination screens, BATCHIE saved 10s of thousands to 100s of thousands of experiments relative to non-adaptive baselines. We conducted a prospective study focusing on pediatric sarcomas with BATCHIE. Our study covered 16 cell lines spanning Ewing sarcoma (EWS), osteosarcoma, rhabdomyosarcoma, as well as non-sarcoma cancers and non-cancer lines. We used a drug library of 206 drugs at two doses. After 15 rounds of BATCHIE-driven data collection, we observed 54K unique combinations, covering 4% of the experimental landscape. On unobserved validation data, the BATCHIE model predictions were highly accurate (Pearson’s rho=0.91, p<10-30) and detected the rare (0.004%) combinations with significant synergy (AUC of ROC=0.85, p<10-5). We further investigated 10 combinations that BATCHIE predicted to have high therapeutic index (TI) for a broad selection of EWS lines, meaning a large differential effect between the predicted viabilities of the control lines and the target lines. We found that the TI scores for the top hits were significantly larger than the rest of the screen (p<10−50), with the median top hit TI score lying in the 98th percentile of observed TI scores. We further validated 6 of the top hits in an ex vivo study on 2 patient-derived EWS samples, again finding significantly large TI values (p<10-13), with the median ex vivo TI score lying in the 96th percentile of observed TI scores. The top hits also exhibited biologically plausible rationales including combining PARP inhibitors with topoisomerase 1 inhibitors and alkylating agents, despite our model utilizing no prior knowledge on the molecular targets of our drug library. Combining PARP inhibitors with topoisomerase 1 inhibitors comprise 3 of the 6 currently open phase II clinical combination trials for EWS. Citation Format: Christopher Tosh, Mauricio Tec, Jessica White, Jeffrey F. Quinn, Glorymar Ibanez Sanchez, Paul Calder, Andrew L. Kung, Filemon S. Dela Cruz, Wesley Tansey. BATCHIE: An active learning platform for scalable combination drug screens [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 901.

Abstract 129: Understanding the molecular drivers that regulate Ewing sarcoma cell fate

Abstract Like other translocation-positive sarcomas, Ewing sarcoma (ES) harbors a pathognomonic fusion protein (FP) capable of widespread epigenetic reprogramming that can lead cells to acquire a high-grade, undifferentiated phenotypic state. To better understand how this might occur, we employed a CRISPR knockout (CRISPR-KO) model of A673, targeting exon 4 of the N-terminus EWSR1 portion of the FP and generated a clonal pool (EWSR1 CRISPR KO). We used single-cell RNA and ATAC sequencing (scMultiome) to identify the transcriptional and chromatin changes after the FP knockout (FP KO). We identified several clusters of ES cell heterogeneity. Since the EWSR1-KO is a pooled population, we used known FP-regulated genes to delineate cells bearing the FP KO from wild-type (FP WT) cells. We observed robust expression of FP-activated genes, PRKCB, and LIPI, in many clusters suggesting regular FP function (FP WT). Conversely, FP-repressed genes, LOX, and IGFBP3 were elevated in a single cluster containing only EWSR1-KO cells. This suggests the FP was successfully knocked out in this cluster (FP KO). That FP KO cluster also lost transcriptional resemblance to ES. A granular identification with cell lines confirmed that all clusters except FP KO were identified as A673 cells. However, the FP KO resembled cell lines with a fibroblastic phenotype. We analyzed enriched motifs and observed no significant difference between the FP KO and FP WT for EWSR1::FLI1. However, the FP KO cells were enriched for motifs in the Fos/Jun family and Smad2/3, which have roles in EMT and mesenchymal differentiation. The FP-KO cells significantly expressed the Fos/Jun family of proteins. Next, we identified peak-gene associations within a ± 500 kb window around the TSS. When we analyzed peaks linked to genes expressed in FP-WT, we found enrichment of the Fos/Jun protein family (p < 1e-34), Ets family, and GGAA-microsatellites. However, the FP-KO were strongly enriched for Fos/Jun protein family (p < 1e-540) with no change in Ets family motif. Knocking out the FP in the A673 cell line significantly shifted the ES lineage. We observed peaks linked to genes positively associated or regulated by the FP were enriched for Fos/Jun family, Ets family, and GGAA-microsatellites. There may be a cooperative interaction between these TFs that affects the genes linked to the FP. The fact that FP loss enriched the Fos/Jun family motifs, but not Ets family or GGAA-microsatellites, suggests that FP loss activates FP-repressed genes and enables chromatin remodeling to open previously inaccessible DNA binding sites. Citation Format: Danh Truong, David McCall, Clement Agyemang, Sandhya Krishnan, Alexander Lazar, Joseph Ludwig. Understanding the molecular drivers that regulate Ewing sarcoma cell fate [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 129.

Abstract 1082: Exploiting lurbinectedin-driven nucleolar relocalization of EWS-FLI1 to develop novel combination therapies for 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.

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.

Abstract 1087: Synergistic mechanisms against pediatric bone sarcoma models: Trabectedin enhances oncolytic virotherapy intratumoral spread and antitumor immune activation

Abstract Osteosarcoma and Ewing sarcoma are the most common pediatric bone malignancies and, despite decades of research, efforts to treat patients with advanced disease remain dismal. To address this inadequacy, we leverage oncolytic herpes simplex virotherapy as a double-edged attack against tumors through 1) tumor cell-specific lysis and 2) antitumor immune stimulation. However, previous trials of oncolytic herpes simplex virotherapy in pediatric sarcomas displayed limited responses, likely due to the observed presence of immunosuppressive monocytes and macrophages. To improve efficacy, we combined oncolytic herpes simplex virotherapy with trabectedin, an FDA-approved DNA-binding agent known to disrupt gene expression and deplete monocytes and macrophages. Surprisingly, we found that the efficacy of this combination far surpassed our predictions, inducing complete and partial regressions in multiple models of Ewing sarcoma and osteosarcoma. To explore potential mechanisms driving this high degree of synergy, we performed single cell RNA sequencing on tumors of mice treated with monotherapy or combination therapy. Interestingly, trabectedin synergized with the oncolytic virotherapy through two unexpected mechanisms. Our analysis revealed that trabectedin increased intratumoral oncolytic virus spread by disrupting expression of the intrinsic antiviral response in human Ewing sarcoma cells. Additionally, trabectedin not only reduced immunosuppressive macrophages but also stimulated cytotoxic granzyme expression in infiltrating T and NK cells in immunocompetent osteosarcoma models. These effector NK and T cells were necessary for the regressions of osteosarcoma allografts treated with trabectedin and oncolytic virotherapy. Thus, trabectedin enhanced both the oncolytic effect of herpes simplex virotherapy and the effector function of cytotoxic lymphocytes that become activated in response to the virus. The combination of trabectedin and oncolytic herpes simplex virotherapy offers the potential to unite these mechanisms against bone tumors to achieve better outcomes for children. Citation Format: Emily Ringwalt, Mark Currier, Andrea Glaspell, Matthew V. Cannon, Chun-Yu Chen, Amy C. Gross, Maren Cam, Pin-Yi Wang, Ryan D. Roberts, Timothy P. Cripe. Synergistic mechanisms against pediatric bone sarcoma models: Trabectedin enhances oncolytic virotherapy intratumoral spread and antitumor immune activation [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 1087.

Abstract 196: In vitro modeling of pediatric solid tumor lung metastases

Abstract Metastasis remains the main cause of death in pediatric cancer patients with the lung being one of the most common affected organs. Our inability to cure these patients is largely due to insufficient knowledge about tumor and metastatic niche plasticity, while existing models are not fully effective in mirroring the response seen in clinical setting. Our goal is to overcome these limitations by establishing in vitro lung metastasis models using tumor/lung organoid co-cultures to study lung infiltration of pediatric solid tumors. We successfully established patient-derived lung organoids from pediatric lung tissue and characterized them according to the presence and the localization of the respective lung epithelial cell types. Co-culture conditions of lung organoids with PDX-derived Ewing sarcoma and osteosarcoma cells were optimized and models established, while the establishment of models for other pediatric solid tumor entities is ongoing. We have observed infiltration of the lung organoids by tumor cells, and our preliminary findings suggest distinct patterns of infiltration among different tumor entities. Furthermore, we have performed single-cell RNA sequencing analysis on these co-cultures in comparison to the parental lung organoids and tumor cells, aiming to uncover the specific signaling pathways activated in the lung metastatic niche in response to tumor cell invasion. Additionally, experiments using conditioned medium suggest that the lung organoid secretome is sufficient to induce homing of osteosarcoma and Ewing sarcoma cells to the organoids, with the CXCL12/CXCR4 signaling axis as a prime candidate driver. To ascertain the extent to which our in vitro model recapitulates the in vivo lung metastatic niche, we performed spatial transcriptomics (using VISIUM/10X Genomics) on formalin-fixed paraffin-embedded (FFPE) tissue blocks of paired primary tumors and lung metastasis samples for Ewing sarcoma and osteosarcoma. Comprehensive data analysis is currently ongoing, which will instruct a stepwise incorporation of additional factors into our modular organoid co-culture model, resulting in a better representation of the lung metastatic niche. Further, the analysis of patient-derived material will elucidate the spatial organization of solid tumor lung metastases and contribute to the identification of signaling pathways implicated in lung infiltration. In conclusion, our study not only unravels the complexities of pediatric solid tumor metastasis, but also lays the foundation for exploring the anti-metastatic potential of pathway-specific compounds targeting tumor/lung interactions using the established in vitro lung metastasis models. This study is supported by grant 35353-B of the Austrian Science Fund. Citation Format: Martha Zylka, Christoph Hafemeister, Lukas Watzke, Ulrike Mann, Didier Surdez, E. Alejandro Sweet-Cordero, Bernadette Liegl-Atzwanger, Matthias Dettmer, Matthias Farlik, Florian Halbritter, Martin Metzelder, Heinrich Kovar, Branka Radic-Sarikas. In vitro modeling of pediatric solid tumor lung metastases [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 196.

Abstract 2851: Characterization of neogenes in desmoplastic small round cell tumors

Abstract Desmoplastic Small Round Cell Tumor (DSRCT) is a rare, soft tissue sarcoma caused by the EWS::WT1 fusion protein (FP). The FP acts as an aberrant transcription factor with oncogenic properties. Recently, oncogenic chimeric transcription factors (OCTF), such as EWS::WT1, were discovered to induce the expression of novel transcripts in otherwise silent genomic regions. These neogenes (NGs) are highly specific to each OCTF and not detected in normal tissue. 37 NGs were found to be associated with the EWS::WT1 FP.We explored the NGs to characterize their expression and regulation in a cohort of MD Anderson Cancer Center patients. We annotated and counted the NGs from the bulk RNA-seq data of 37 specimens. After fusion detection, only 22/37 specimens harbored the EWS::WT1 FP. We found that only the same 22 specimens robustly expressed DSRCT-specific NGs. Interestingly the NGs were heterogeneously expressed, suggesting that there may be patient-specific differences. Single-nucleus RNA-sequencing (snRNA-seq) of Ewing sarcoma (ES), CIC::DUX4 sarcoma (CDS), DSRCT, and osteosarcoma revealed that only the three sarcomas driven by an OCTF robustly expressed NGs. Notably, the DSRCT-specific NGs were only expressed in DSRCT. Similarly, ES and CDS only expressed their subtype-specific NGs. Given the robust expression of the DSRCT-specific NGs in DSRCT, we asked if the EWS::WT1 FP regulated the expression of the NGs. Knockdown (KD) of the EWS::WT1 FP in four DSRCT cell lines showed a consistent reduction in DSRCT-specific NGs. While there was variability in NG expression and EWS::WT1 FP KD sensitivity, we identified seven NGs decreased by at least 75% in all four cell lines and an additional eight NGs with 75% reduction across three cell lines. BOD-DSRCT and SK-DSRCT2 had the largest number of NGs reduced after KD, with 25/37 and 20/37 NGs with 75% expression reduction, respectively. EWS::WT1 FP has two known isoforms generated by alternative splicing in the WT1 domain that includes or excludes three amino acids, lysine, threonine, and serine (KTS) between zinc fingers 3 and 4, producing E+KTS and E-KTS isoforms. By transducing one or both isoforms into the mesothelial cell line LP9, we found 3/37 NGs were induced by the E+KTS isoform and 17/37 were induced by the E-KTS isoform, suggesting that the E-KTS isoform is primarily responsible for NG expression. Current work is underway investigating the relationship between EWS::WT1 FP binding and NG expression and the potential for NG to produce novel peptides that could serve as DSRCT immunotherapy targets. Citation Format: Danh Truong, Justin Magrath, Sean Lee, Joseph Ludwig. Characterization of neogenes in desmoplastic small round cell 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 2851.

Intraoperative radiation therapy for pediatric sarcomas and other solid tumors.

To evaluate local failure (LF) and toxicity after intraoperative radiation therapy (IORT) in pediatric solid tumors (ST). A single-institution retrospective study of 96 pediatric patients (108 applications) with ST treated from 1995 to 2022 with IORT. LF was calculated via cumulative incidence function and overall survival (OS) by Kaplan-Meier method, both from the day of surgery. Median age at time of IORT was 8years (range: 0.8-20.9years). Median follow-up for all patients and surviving patients was 16months and 3years, respectively. The most common histologies included rhabdomyosarcoma (n=42), Ewing sarcoma (n=10), and Wilms tumor (n=9). Most (95%) received chemotherapy, 37% had prior external beam radiation therapy to the site of IORT, and 46% had a prior surgery for tumor resection. About half (54%) were treated with upfront IORT to the primary tumor due to difficult circumstances such as very young age or challenging anatomy. The median IORT dose was 12Gy (range: 4-18Gy), and median area treated was 24cm2 (range: 2-198cm2). The cumulative incidence of LF was 17% at 2years and 23% at 5years. Toxicity from IORT was reasonable, with postoperative complications likely related to IORT seen in 15 (16%) patients. Our study represents the largest and most recent analysis of efficacy and safety of IORT in pediatric patients with ST. Less than one quarter of all patients failed locally with acceptable toxicities. Overall, IORT is an effective and safe technique to achieve local control in patients with challenging circumstances.

Abstract 4392: Neuropeptide Y system in glioblastoma

Abstract Gliomas constitute the most common primary cerebral tumors of the central nervous system (CNS), with glioblastoma (GBM) representing the most aggressive subtype. Neurotransmitters are important players in maintaining physiological processes in CNS and abnormal levels of their expression is pathogenic, leading to different processes, including neoplasia. Neuropeptide Y (NPY) is one of the neurotransmitters that is found to play a pivotal role in several malignancies. NPY, acting via its cognate receptors Y1, Y2 and Y5 (Y1R, Y2R and Y5R), has been shown to stimulate cancer cell proliferation, migration, invasiveness and dissemination, as well as tumor vascularization in various malignancies including neuroblastoma, Ewing sarcoma, colon, breast, prostate and liver cancers. Thus, the goal of this study was to determine if the NPY system is involved in glioma biology. Our immunohistochemistry staining of NPY, Y1R, Y2R, and Y5R in human GBM tissues revealed positive staining for NPY and Y2R in GBM cells. No expression of Y1R and Y5R was detected in GBM neoplastic population. To further elucidate the association between NPY and its receptors with GBM tumor stage and patients' survival, we used the available clinical data from the Cancer Genome Atlas Program (TCGA) database to analyze the gene expression of the NPY system. Y2R expression was increased in high-grade gliomas, while mRNA levels of NPY, Y1R and Y5R inversely correlated with the degree of glioma malignancy. Moreover, high Y2R expression was associated with worse survival in GBM patients. Importantly, endothelial Y2R is also responsible for angiogenic effects of NPY, suggesting its role in GBM vascularization. Altogether, these data suggest the involvement of NPY/Y2R axis in glioma progression toward more aggressive phenotype. Further functional studies are required to determine the role of the Y2R axis in this process. Citation Format: Nouran Abualsaud, Jason Tilan, Bahauddeen M. Alrfaei, Harrison Glor, Taylor Polk, Ewa Izycka-Swieszewska, Joanna Kitlinska. Neuropeptide Y system in glioblastoma [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 4392.