Osteosarcoma (OS) is one of the most common and a highly aggressive bone cancer in children. It typically occurs during childhood or adolescence and often develops in areas of bone growth. Standard front-line therapy achieves cure rate of 60-70% for non-metastatic OS and merely a 30% for metastatic OS. Unfortunately, the treatment of OS patients has not significantly improved in recent decades, in particular for relapsed OS, presenting an unmet clinical need. We performed advanced mass spectrometry (MS)-based proteomic profiling and functional investigations to identify novel drug targets. We proteome profiled a cohort of 154 samples comprising OS tissue samples from relapsed patients of the INFORM cohort, cell lines and healthy controls. Using our high-throughput proteomic platform, we quantified a total of >12,100 proteins by DIA-PASEF with exceptional reproducibility. In addition, we have performed RNAseq, WES, EPIC array and Phosphoproteome for most of these samples to allow a multiomics examination.

Our analysis reveals that one of the proteomic clusters enriched for MYC targets, RNA processing and splicing in OS patients significantly associates with low overall survival, and displays a higher rate of TP53 mutation as well as MYC amplifications. A similar cluster could be identified in OS cell lines, indicating a so far undescribed OS subgroup and enabling functional validations. In line with the observation of lower overall survival in patients, cell lines falling into the respective subgroup show higher proliferation, underlining the increased aggressiveness of this subgroup. Further we found overexpression and hyperactivation of MYC is not associated with MYC amplification. Interestingly, no difference in phosphorylation status of the key regulating MYC Phosphosite S77 could be detected between MYC amplified and MYC non-amplified patients, strongly suggesting that MYC overexpression and hyperactivation is independent of the MYC amplification status.

Importantly, several pan-spliceosomal inhibitors do not show differential activity between the OS subgroups, indicating existence of specific functional dependencies. We identified three splicing proteins to be upregulated in the respective patient and cell line subgroups that are more essential for the respective OS cell lines compared to cell lines from the other subgroup using the Depmap database. Additionally, overexpression of these three proteins correlates with low event-free survival in four publicly available datasets. Ongoing efforts in our lab aim at exploring the functional roles played by these proteins using CRISPR/Cas9 genome editing as well as knock-down and overexpression approaches.

In summary, we present an unsupervised proteogenomic clustering of relapsed Osteosarcoma which reveals specific vulnerabilities in a subset of Osteosarcoma patients. These findings will help to further develop therapeutic options for patients showing bad clinical prognostic characteristics.