Unravelling HRDness and therapeutic opportunities in soft tissue and bone sarcoma

Genomic instability is a hallmark of many cancers. Aberrant proliferation in cancer cells leads to the accumulation of alterations in genes that belong to the homologous recombination (HR) DNA double-strand break (DSB) repair pathway. Deficiency in HR-mediated repair (HRR) exacerbates genomic instability and correlates with poor prognosis and development of metastases. Determining HRR deficiency (HRD) in a given tumour is of major clinical relevance as it is associated with therapeutic vulnerabilities. HRD has been widely investigated in certain tumor types such as ovarian and breast cancer but remains greatly unexplored in sarcoma, a rare and heterogeneous group of mesenchymal cancer. Here, we show that specific sarcoma entities are characterized by high levels of genomic instability signatures and a wide range of molecular alterations in HRR genes, while exhibiting a complex pattern of chromosomal instability features. Furthermore, sarcomas carrying HRDness traits exhibit a distinct SARC-HRD transcriptional signatures that predicts PARP inhibitor sensitivity in patient-derived ex vivo sarcoma models. Concomitantly, HRDhigh sarcoma cell models lack RAD51 nuclear foci formation upon DNA damage, further evidencing defects in HR-mediated DNA repair. We further identify the WEE1 kinase as a therapeutic vulnerability for sarcomas with HRDness traits and demonstrate clinical benefit of combining DNA damaging agents and inhibitors of DNA repair pathways in patient-derived ex vivo cell models and in a leiomyosarcoma patient. In summary, we provide the most comprehensive analysis of HRDness in sarcoma to date and support a bench-to-bedside personalized oncological approach to successfully treat sarcoma patients. RNA from patient-derived sarcoma cell models was extracted using the Maxwell RSC simplyRNA Tissue kit (Promega AS1340). The extracted RNA was quantified with a Nanodrop (ThermoFisher Scientific) and the High Sensitivity RNA ScreenTape Assay for TapeStation systems (Agilent 5067). RNA-sequencing libraries were prepared using the Illumina Stranded mRNA sample preparation protocol following the manufacturer’s instructions. Each library was sequenced in paired-end mode using the NovaSeq 6000 platform and resulting reads were mapped to the human transcriptome. Differential gene expression between HRDhigh and HRDlow sarcoma samples from the TCGA cohort was investigated by downstream processing of raw counts using the R package DESeq2 26.