RNA sequencing and functional studies of patient-derived cells reveal that neurexin-1 and regulators of this pathway are associated with poor outcomes in Ewing sarcoma

Michael Parry,Alastair Droop,Lee Jeys,Elizabeth Ann Roundhill,Kenneth S Rankin,Susan Ann Burchill,Mariona Chicon-Bosch

doi:10.1007/s13402-021-00619-8

Abstract

PurposeThe development of biomarkers and molecularly targeted therapies for patients with Ewing sarcoma (ES) in order to minimise morbidity and improve outcome is urgently needed. Here, we set out to isolate and characterise patient-derived ES primary cell cultures and daughter cancer stem-like cells (CSCs) to identify biomarkers of high-risk disease and candidate therapeutic targets.MethodsThirty-two patient-derived primary cultures were established from treatment-naïve tumours and primary ES-CSCs isolated from these cultures using functional methods. By RNA-sequencing we analysed the transcriptome of ES patient-derived cells (n = 24) and ES-CSCs (n = 11) to identify the most abundant and differentially expressed genes (DEGs). Expression of the top DEG(s) in ES-CSCs compared to ES cells was validated at both RNA and protein levels. The functional and prognostic potential of the most significant gene (neurexin-1) was investigated using knock-down studies and immunohistochemistry of two independent tumour cohorts.ResultsES-CSCs were isolated from all primary cell cultures, consistent with the premise that ES is a CSC driven cancer. Transcriptional profiling confirmed that these cells were of mesenchymal origin, revealed novel cell surface targets for therapy that regulate cell-extracellular matrix interactions and identified candidate drivers of progression and relapse. High expression of neurexin-1 and low levels of regulators of its activity, APBA1 and NLGN4X, were associated with poor event-free and overall survival rates. Knock-down of neurexin-1 decreased viable cell numbers and spheroid formation.ConclusionsGenes that regulate extracellular interactions, including neurexin-1, are candidate therapeutic targets in ES. High levels of neurexin-1 at diagnosis are associated with poor outcome and identify patients with localised disease that will relapse. These patients could benefit from more intensive or novel treatment modalities. The prognostic significance of neurexin-1 should be validated independently.

Highlights

Ewing sarcoma (ES) is a tumour of bone and soft tissues, commonly arising in young people aged 10–25 years [1]
30–40% of patients with localised tumours will develop multi-drug resistant (MDR) disease leading to relapse and poor outcomes typically associated with widespread metastatic disease, with 5 year survival of less than 10% [6]
All cultures (32/32) contained a translocation involving the Ewing sarcoma breakpoint region 1 (EWSR1) gene from chromosome 22q12 detected by Fluorescence in situ hybridisation (FISH) and/or reverse transcriptase polymerase chain reaction (RT-PCR)

Summary

Introduction

Ewing sarcoma (ES) is a tumour of bone and soft tissues, commonly arising in young people aged 10–25 years [1]. Twenty-five percent of patients present with metastatic disease, most frequently in multiple sites including bone/bone marrow/lungs or lungs only. These patterns of metastasis are associated with overall survival rates of 10–20% and 40%, respectively [2]. 30–40% of patients with localised tumours will develop multi-drug resistant (MDR) disease leading to relapse and poor outcomes typically associated with widespread metastatic disease, with 5 year survival of less than 10% [6]. For all patients that survive therapy, 1 in 10 will relapse up to 20 years after initial diagnosis [7], many experiencing treatment-induced morbidity [8,9,10]. The development of molecularly targeted therapies to minimise morbidity and improve outcomes for patients with ES are urgently needed

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Conclusion