Molecular characterization of osteosarcoma and Ewing sarcoma in pediatric patients reveals novel RNA-based intervention points

Ewing sarcoma (EW) and osteosarcoma (OS) are the two most common bone sarcomas (BS) in the pediatric population. BS are heterogeneous diseases characterized by distinct clinical and pathologic features. The high metastasis recurrence and extremely poor clinical prognosis pose the urgent need for new treatment strategies for these young patients. Within the national clinical trial SAR_GEN-ITA (ClinicalTrial.gov id:NCT04621201), we aimed at identifying novel putative targets that can be exploited for immunotherapy. Whole exome (WXS) and total RNA sequencing (RNA-seq) of 28 OS and 18 EW samples from pediatric patients were performed. WXS data were exploited as previously described. In particular, predisposing and somatic mutations and copy number variations were identified. A differential gene expression analysis was performed using Deseq2 R package. Then, using available RNA-seq of osteoblast samples as controls, rMATS was applied to outline the alternative splicing landscape of OS and EW. Trans- and cis-acting regulatory elements were identified combining RNAmotifs results with mCross information and eCLIP data from ENCODE. Novel EW and OS splice junctions were further inspected for the identification of neoepitopes using ISOTOPE. At the genomic level, we confirmed the low mutational burden and high genomic instability of these BSs. At the transcriptomic level, the differential gene expression analysis revealed a significant overexpression of genes that contribute to epithelial to mesenchymal transition (EMT) and are targets of KRAS in OS and EW samples, respectively. We further challenged RNA-seq data to identify alternative splicing (AS) events that are differentially included upon cancer insurgence. We identified 4,025 and 3,890 differentially (AS) events in OS and EW respectively. Through a motif analysis we found factors, such as SRSF1 and U2AF1, responsible for the somatic rewiring of the AS landscape of BS. We then focused on nonsense mediated-determinant exons, whose inclusion has been related to tumorigenesis, namely poison and essential exons. Amongst other oncogenic splicing events, the inclusion of the micro-exon 30 of the cancer gene FLNA emerged as the strongest candidate in both OS and EW samples. Furthermore, since AS events are a source of neoantigens that are potentially presented by MHC-I complexes to a greater extent than single point mutations, we inspected the tumor-specific splicing junctions. We found 454 novel junctions that gave rise to 2,755 neoepitopes, most of them being private to patients. Nevertheless, we identified seven events shared by OS and EW. Among them, we selected the XPO1ex4-8 event, for belonging to a cancer-related and essential gene in OS and EW. In line with previous works we identified low mutational burden and high heterogeneity between patients. Heterogeneity is known for strongly impacting on cancer progression and drug response. Exploring RNA-seq data, we retrieved a deregulation in KRAS signaling and EMT. Notably, the overexpression of an EMT component has been recently associated with the increased aggressiveness of pediatric sarcomas. By integrating analyses of cis and trans-acting elements we proposed that the BS splicing landscape is orchestrated by SRSF1 and U2AF1. We found that several cancer-related exons were differentially included upon cancer insurgence. Of these, we proposed FLNA ex.30 as a possible target. Additionally, we identified XPO1 ex.4-8 as a neoepitope event rising from a novel junction retrieved both in OS and EW. Together, our findings revealed novel candidate intervention points for lowly mutated pediatric BS tumors that could be exploited with RNA-based approaches and immunotherapies.