Exploiting the Stemness and Chemoresistance Transcriptome of Ewing Sarcoma to Identify Candidate Therapeutic Targets and Drug-Repurposing Candidates

SIMPLE SUMMARY: Ewing sarcoma is a cancer arising most frequently in teenagers and young adults. For many patients, outcomes are the same today as they were 30 years ago, emphasising the need for more effective treatments to eradicate the cells responsible for progression and relapse. These cells responsible for progression and relapse have been identified using assays that evaluate functional characteristics and expression of cell surface markers. For the first time, we reveal ABCG1 as an additional potential cell surface marker of progression. In rare cancers like Ewing sarcoma, commercial development of new drugs is seldom a priority, reflecting the small number of patients and lack of well-characterised molecular subtypes. Therefore, rather than creating new drugs, which can take 20 to 30 years, repurposing of existing drugs may be an efficient cost-effective strategy to accelerate novel molecularly targeted therapy into clinical trials for patients with Ewing sarcoma. To identify candidate molecular targets, we have used a combination of functional assays and transcriptomic analyses to characterize the cells responsible for progression and relapse. We have then applied a bespoke in silico pipeline to find drugs with known safety profiles that bind to these targets. In the future, after preclinical validation of efficacy and specificity in Ewing sarcoma, some of these drugs may be assessed as combination treatments in clinical trials, with the goal of improving outcomes. ABSTRACT: Outcomes for most patients with Ewing sarcoma (ES) have remained unchanged for the last 30 years, emphasising the need for more effective and tolerable treatments. We have hypothesised that using small-molecule inhibitors to kill the self-renewing chemotherapy-resistant cells (Ewing sarcoma cancer stem-like cells; ES-CSCs) responsible for progression and relapse could improve outcomes and minimise treatment-induced morbidities. For the first time, we demonstrate that ABCG1, a potential oncogene in some cancers, is highly expressed in ES-CSCs independently of CD133. Using functional models, transcriptomics and a bespoke in silico drug-repurposing pipeline, we have prioritised a group of tractable small-molecule inhibitors for further preclinical studies. Consistent with the cellular origin of ES, 21 candidate molecular targets of pluripotency, stemness and chemoresistance were identified. Small-molecule inhibitors to 13 of the 21 molecular targets (62%) were identified. POU5F1/OCT4 was the most promising new therapeutic target in Ewing sarcoma, interacting with 10 of the 21 prioritised molecular targets and meriting further study. The majority of small-molecule inhibitors (72%) target one of two drug efflux proteins, p-glycoprotein (n = 168) or MRP1 (n = 13). In summary, we have identified a novel cell surface marker of ES-CSCs and cancer/non-cancer drugs to targets expressed by these cells that are worthy of further preclinical evaluation. If effective in preclinical models, these drugs and drug combinations might be repurposed for clinical evaluation in patients with ES.