Inducing Mitotic Catastrophe as a Therapeutic Approach to Improve Outcomes in Ewing Sarcoma.

Ewing sarcoma (EWS) is an aggressive pediatric malignancy of the bone and soft tissues in need of novel therapeutic options. To identify potential therapeutic targets, we focused on essential biological pathways that are upregulated by EWS-FLI1, the primary oncogenic driver of EWS, including mitotic proteins such as Aurora kinase A (AURKA) and kinesin family member 15 (KIF15) and its binding partner, targeting protein for Xklp2 (TPX2). KIF15/TPX2 cooperates with KIF11, a key mitotic kinesin essential for mitotic spindle orientation. Given the lack of clinical-grade KIF15/TPX2 inhibitors, we chose to target KIF11 (using SB-743921) in combination with AURKA (using VIC-1911) given that phosphorylation of KIF15S1169 by Aurora A is required for its targeting to the spindle. In vitro, the drug combination demonstrated strong synergy (Bliss score ≥ 10) at nanomolar doses. Colony formation assay revealed significant reduction in plating efficiency (1-3%) and increased percentage accumulation of cells in the G2/M phase with the combination treatment (45-52%) upon cell cycle analysis, indicating mitotic arrest. In vivo studies in EWS xenograft mouse models showed significant tumor reduction and overall effectiveness: drug combination vs. vehicle control (p ≤ 0.01), SB-743921 (p ≤ 0.01) and VIC-1911 (p ≤ 0.05). Kaplan-Meier curves demonstrated superior overall survival with the combination compared to vehicle or monotherapy arms (p ≤ 0.0001).

Identification of small extracellular vesicle protein biomarkers for pediatric Ewing Sarcoma.

Ewing Sarcoma (EWS) is the second most common osseous malignancy in children and young adults after osteosarcoma, while it is the fifth common osseous malignancy within adult age population. The clinical presentation of EWS is quite often non-specific, with the most common symptoms at presentation consisting of pain, swelling or general discomfort. The dearth of clinically relevant diagnostic or predictive biomarkers continues to remain a pressing clinical challenge. Identification of tumor specific biomarkers can lend towards an early diagnosis, expedited initiation of therapy, monitoring of therapeutic response, and early detection of recurrence of disease. We carried-out a complex analysis of cell lines and cell line derived small extracellular vesicles (sEVs) using label-free-based Quantitative Proteomic Profiling with an intent to determine shared and distinct features of these tumor cells and their respective sEVs. We analyzed EWS cells with different EWS-ETS fusions (EWS-FLI1 type I, II, and III and EWS-ERG) and their corresponding sEVs. Non-EWS controls included osteosarcoma, rhabdomyosarcoma, and benign cells, i.e., osteoid osteoma and mesenchymal stem cells. Proteomic profiling identified new shared markers between cells and their corresponding cell-derived sEVs and markers which were exclusively enriched in EWS-derived sEVs. These exo-biomarkers identified were validated by in silico approaches of publicly available protein databases and by capillary electrophoresis based western analysis (Wes). Here, we identified a protein biomarker named UGT3A2 and found its expression highly specific to EWS cells and their sEVs compared to control samples. Clinical validation of UGT3A2 expression in patient tumor tissues and plasma derived sEV samples demonstrated its specificity to EWS, indicating its potential as a EWS biomarker.

MicroRNA Content of Ewing Sarcoma Derived Extracellular Vesicles Leads to Biomarker Potential and Identification of a Previously Undocumented EWS-FLI1 Translocation.

Objective: Ewing Sarcoma Family of Tumors (ESFT) are a highly aggressive pediatric bone and soft tissue malignancy with poor outcomes in the refractory and recurrent setting. Over 90% of Ewing Sarcoma (ES) tumors are driven by the pathognomonic EWS-ETS chimeric transcripts and their corresponding oncoproteins. It has been suggested that the EWS-ETS oncogenic action can mediate microRNA (miRNA) processing. Importantly, small extracellular vesicles (sEVs), including those frequently referred to as exosomes have been shown to be highly enriched with tumor-derived small RNAs such as miRNAs. We hypothesized that ESFT-specific sEVs are enriched with certain miRNAs which could be utilized toward an exo-miRNA biomarker signature specific to this disease. Methods: We performed miRNAseq to compare both the exo-derived and cell-derived miRNA content from 8 ESFT, 2 osteosarcoma, 2 non-cancerous cell lines, and pediatric plasma samples. Results: We found that sEVs derived from ESFT cells contained nearly 2-fold more number of unique individual miRNAs as compared to non-ESFT samples. Quantitative analysis of the differential enrichment of sEV miRNAs resulted in the identification of 62 sEV-miRNAs (exo-miRNAs) with significant (P < .05) enrichment variation between ESFT and non-ESFT sEV samples. To determine if we could utilize this miRNA signature to diagnose ESFT patients via a liquid biopsy, we analyzed the RNA content of total circulating sEVs isolated from 500 µL plasma from 5 pediatric ESFT patients, 2 pediatric osteosarcoma patients, 2 pediatric rhabdomyosarcoma patients, and 4 non-cancer pediatric controls. Pearson's clustering of 60 of the 62 candidate exo-miRNAs correctly identified 80% (4 of 5) of pathology confirmed ESFT patients. Importantly, RNAseq analysis of tumor tissue from the 1 outlier, revealed a previously uncharacterized EWS-FLI1 translocation.Conclusions: Taken together, these findings support the development and validation of an exo-miRNA-based liquid biopsy to aid in the diagnosis and monitoring of ESFT.

Ewing sarcoma family of tumors-derived small extracellular vesicle proteomics identify potential clinical biomarkers.

PURPOSE: Ewing Sarcoma Family of Tumors (ESFT), the second most common pediatric osseous malignancy, are characterized by the pathognomonic chromosomal EWS-ETS translocation. Outside of tumor biopsy, no clinically relevant ESFT biomarkers exist. Additionally, tumor burden assessment at diagnosis, monitoring of disease responsiveness to therapy, and detection of disease recurrence are limited to radiographic imaging. To identify new, clinically relevant biomarkers we evaluated the proteome of a subset of ESFT-derived small extracellular vesicles (sEVs). MATERIALS AND METHODS: We performed the first high quality proteomic study of ESFT-derived sEVs from 5 ESFT cell lines representing the most common EWS-ETS fusion types and identified 619 proteins composing the core ESFT sEV proteome. We compared these core proteins to databases of common plasma-based proteins and sEV-associated proteins found within healthy plasma to identify proteins unique or enriched within ESFT. RESULTS: From these analyses, two membrane bound proteins with biomarker potential were selected, CD99/MIC2 and NGFR, to develop a liquid-based assay enriching of ESFT-associated sEVs and detection of sEV mRNA cargo (i.e., EWS-ETS transcripts). We employed this immuno-enrichment approach to diagnosis of ESFT utilizing plasma (250 µl) from both localized and metastatic ESFT pediatric patients and cancer-free controls, and showed significant diagnostic power [AUC = 0.92, p = 0.001 for sEV numeration, with a PPV = 1.00, 95% CI = (0.63, 1.00) and a NPV = 0.67, 95% CI = (0.30, 0.93)]. CONCLUSIONS: In this study, we demonstrate utilization of circulating ESFT-associated sEVs in pediatric patients as a source of minimally invasive diagnostic and potentially prognostic biomarkers.

Beyond tumor mutational burden: potential and limitations in using exosomes to predict response to immunotherapy.

Introduction: Immune checkpoint blockade (ICB) has ushered in a new era of cancer therapeutics. The standard for determining which patients might benefit from ICB-based therapies is through the assessment of tumor mutational burden using formalin-fixed paraffin-embedded (FFPE) tumor tissue samples; however, this strategy is imperfect. The discovery of exosomal PD-L1 has placed these nano-vesicles to the forefront of immunotherapy biomarker development. Exosomes and other extracellular vesicles contain proteins and nucleic acids specific to their cell of origin and their production is increased in disease state such as cancer and can be isolated from most types of liquid biopsy. Given this opportunity, a large-scale bioengineering effort has centered on developing technologies capable of isolating distinct subsets of exosomes and interrogating their content for biomarker discovery.Areas covered: This review investigates the current state of small extracellular vesicles (sEVs), focusing on exosomes, as they relate to biomarkers of IBC. We will discuss technologies being developed to both capture and evaluate exosomal cargo and as some of the challenges they face.Expert opinion: The advancement of microfluidic technologies, along with rapidly evolving methodologies in RNAseq and proteomics, are making the potential of utilizing exosomes as prognostic and diagnostic biomarkers of ICB into a likely reality.

Targeted inhibition of histone deacetylase leads to suppression of Ewing sarcoma tumor growth through an unappreciated EWS-FLI1/HDAC3/HSP90 signaling axis.

Ewing sarcoma (ES) are aggressive pediatric bone and soft tissue tumors driven by EWS-ETS fusion oncogenes, most commonly EWS-FLI1. Treatment of ES patients consists of up to 9 months of alternating courses of 2 chemotherapeutic regimens. Furthermore, EWS-ETS-targeted therapies have yet to demonstrate clinical benefit, thereby emphasizing a clinical responsibility to search for new therapeutic approaches. Our previous in silico drug screening identified entinostat as a drug hit that was predicted to reverse the ES disease signatures and EWS-FLI1-mediated gene signatures. Here, we establish preclinical proof of principle by investigating the in vitro and in vivo efficacy of entinostat in preclinical ES models, as well as characterizing the mechanisms of action and in vivo pharmacokinetics of entinostat. ES cells are preferentially sensitive to entinostat in an EWS-FLI1 or EWS-ERG-dependent manner. Entinostat induces apoptosis of ES cells through G0/G1 cell cycle arrest, intracellular reactive oxygen species (ROS) elevation, DNA damage, homologous recombination (HR) repair impairment, and caspase activation. Mechanistically, we demonstrate for the first time that HDAC3 is a transcriptional target of EWS-FLI1 and that entinostat inhibits growth of ES cells through suppressing a previously unexplored EWS-FLI1/HDAC3/HSP90 signaling axis. Importantly, entinostat significantly reduces tumor burden by 97.4% (89.5 vs. 3397.3 mm3 of vehicle, p < 0.001) and prolongs the median survival of mice (15.5 vs. 8.5 days of vehicle, p < 0.001), in two independent ES xenograft mouse models, respectively. Overall, our studies demonstrate promising activity of entinostat against ES, and support the clinical development of the entinostat-based therapies for children and young adults with metastatic/relapsed ES. KEY MESSAGES: • Entinostat potently inhibits ES both in vitro and in vivo. • EWS-FLI1 and EWS-ERG confer sensitivity to entinostat treatment. • Entinostat suppresses the EWS-FLI1/HDAC3/HSP90 signaling. • HDAC3 is a transcriptional target of EWS-FLI1. • HDAC3 is essential for ES cell viability and genomic stability maintenance.

Ultrasensitive quantification of tumor mRNAs in extracellular vesicles with an integrated microfluidic digital analysis chip.

Extracellular vesicles (EVs) present a promising liquid biopsy for cancer diagnosis. However, it remains a daunting challenge to quantitatively measure molecular contents of EVs including tumor-associated mRNAs. Herein, we report a configurable microwell-patterned microfluidic digital analysis platform combined with a dual-probe hybridization assay for PCR-free, single-molecule detection of specific mRNAs in EVs. The microwell array in our device is configurable between the flow-through assay mode for enhanced hybridization capture and tagging of mRNAs and the digital detection mode based on femtoliter-scale enzymatic signal amplification for single-molecule counting of surface-bound targets. Furthermore, a dual-probe hybridization assay has been developed to enhance the sensitivity of the digital single-molecule detection of EV mRNAs. Combining the merits of the chip design and the dual-probe digital mRNA hybridization assay, the integrated microfluidic system has been demonstrated to afford quantitative detection of synthetic GAPDH mRNA with a LOD as low as 20 aM. Using this technology, we quantified the level of GAPDH and EWS-FLI1 mRNAs in EVs derived from two cell lines of peripheral primitive neuroectodermal tumor (PNET), CHLA-9 and CHLA-258. Our measurements detected 64.6 and 43.5 copies of GAPDH mRNA and 6.5 and 0.277 copies of EWS-FLI1 fusion transcripts per 105 EVs derived from CHLA-9 and CHLA-258 cells, respectively. To our knowledge, this is the first demonstration of quantitative measurement of EWS-FLI1 mRNA copy numbers in Ewing Sarcoma (EWS)-derived EVs. These results highlight the ultralow frequency of tumor-specific mRNA markers in EVs and the necessity of developing highly sensitive methods for analysis of EV mRNAs. The microfluidic digital mRNA analysis platform presented here would provide a useful tool to facilitate quantitative analysis of tumor-associated EV mRNAs for liquid biopsy-based cancer diagnosis and monitoring.

Molecular assessment of circulating exosomes toward liquid biopsy diagnosis of Ewing sarcoma family of tumors.

Ewing sarcoma was first described in 1921 in the Proceedings of the New York Pathological Society by an eminent American pathologist from Cornell named James R. Ewing as a "diffuse endothelioma of bone." Since this initial description, more has been discovered regarding Ewing sarcoma and in the 1980's both Ewing sarcoma and peripheral primitive neuroectodermal tumors due to their similar features and shared identical genetic abnormality were grouped into a class of cancers entitled Ewing sarcoma family of tumors (ESFTs). Ewing sarcoma is the second most common pediatric osseous malignancy followed by osteosarcoma, with highest incidence among 10-20 years old. Ewing sarcoma is consistently associated with chromosomal translocation and functional fusion of the EWSR1 gene to any of several structurally related transcription factor genes of the E26 transformation-specific family. These tumor-specific molecular rearrangements are useful for primary diagnosis, may provide prognostic information, and present potential therapeutic targets. Therefore, ways to rapidly and efficiently detect these defining genomic alterations are of clinical relevance. Within the past decade, liquid biopsies including extracellular vesicles (EVs), have emerged as a promising alternative and/or complimentary approach to standard tumor biopsies. It was recently reported that fusion mRNAs from tumor-specific chromosome translocations can be detected in Ewing sarcoma cell-derived exosomes. Within this review, we overview the current advances in Ewing sarcoma and the opportunities and challenges in exploiting circulating exosomes, primarily small bioactive EVs (30-180 nm), as developing sources of biomarkers for diagnosis and therapeutic response monitoring in children and young adult patients with ESFT.