Validation of Driver Genetic Aberrations in Undifferentiated Pleomorphic Sarcoma: Focus on the Role of YAP1 Utilising a CRISPR/Cas9 Approach.

Soft tissue sarcoma (STS) is a rare, diverse group of tumours that are characterised by genetic, pathological and clinical diversity. STS comprise less than 1% of all adult malignancies and consist of more than 100 histological and molecular subtypes. Undifferentiated pleomorphic sarcoma (UPS) is a sarcoma with no specific line of differentiation and a pleomorphic morphology. Although UPS is characterised by complex genetic abnormalities with numerous copy number aberrations, none of these abnormalities are specific to UPS. In this study, three amplified genes, previously identified by our research team as having a potential driver role in UPS tumorigenesis, were functionally analyzed to establish a correlation between the gene expression and the aberrant copy number. Initially, semi-quantitative analysis, by immunocytochemistry and western blotting were performed for YAP1, VGLl3 and JUN. Based on these analyses, one of these genes, YAP1 was chosen for further study. The effects of YAP1 inhibition on the UPS cell lines, was investigated using a small molecule inhibitor (CA3). The inhibition of YAP1 resulted in a significant reduction of the proliferation of the UPS cells. Moreover, such inhibition resulted in inducing apoptosis in these cells. In the light of these finding, Crispr/Cas9 technology was successfully undertaken on the four UPS cell lines to knock out YAP1. The knockout efficiency was assessed by different approaches, Genomic cleavage detection (GCD) assay, tracking indels by decomposition (TIDE) and Inference of CRISPR Edits (ICE). Consequently, single cell colonies were derived from each cell line to obtain cells with homogeneously edited genomes. For these clones, the impacts of Crispr/Cas9 editing system were evaluated for both mRNA and protein levels. The proliferation, migration and invasion of these clones and their wild-type correspondent were therefore compared to investigate the impact of YAP1 knockout on the development and progression of UPS. Collectively, the findings in this present study suggest a driver role of YAP1 in the UPS tumorigenesis and provide a plausible therapeutic target for UPS treatment.