Hippo pathway targeting in TP53-mutant Uterine Leiomyosarcoma

Introduction. Uterine Leiomyosarcoma (uLMS)is a rare malignant neoplasm accounting for ~1% of female genital tumors. uLMS is characterized by an aggressive clinical course and a scarce response to chemotherapy. The high frequency ofTP53inactivation (>60% of patients) is linked to uLMS progression and therapy resistance. Recent evidence prove that the Hippo signaling cooperates with wild-type p53 as tumor suppressor to induce senescence and apoptosis in response to stress conditions, while mutant p53 forms a complex with YAP promoting the transcription of key cell cycle regulators. Thus, we explored Hippo pathway deregulation inTP53-mutated uLMS and in gene-edited induced Pluripotent Stem Cells (iPSC) differentiated towards the mesodermal layer. Material and Methods. The effects of the YAP inhibitor Verteporfin (VP) on cell viability, proliferation and apoptosis were measured in twoTP53-mutated uLMS cell lines (SK-UT-1 and SK-LMS-1) by MTT assay, Brdu Cell Profliferation assay and cytometry by Annexin-V kit, respectively. Deregulation of the Hippo signaling was analyzed by quantitative PCR on the main readouts of the pathway (CTGF and Cyr61). RNA sequencing was performed with the Illumina Truseq mRNA stranded kit. VP-treatment was performed onTP53-/-iPSC clones, previously obtained byTP53-knockout genome editing and then differentiated towards the mesodermal layer. Results and Discussions. VP treatment induced cell viability decrease on both SK-UT-1 (IC50=3,4 uM) and SK-LMS-1 (IC50=3,7 uM), and resulted in reduced cell proliferation, increased percentage of apoptotic cells and decreased expression of the YAP-target genes. Gene expression profiling of VP-treated cell lines showed an upregulation of the p53 pathway and a downregulation of genes involved in myogenesis and epithelial to mesenchymal transition. Further, pluripotent and differentiatedTP53-/-iPSC clones showed upregulation of the Hippo pathway readouts with respect to their wild-type counterpart and displayed a viability decrease upon VP treatment with an IC50of 1,3 uM. Conclusion. These results prove that interfering with Hippo pathway deregulation inTP53-mutated uLMS could represent a novel approach for an aggressive disease orphan of effective therapies. In this context, we demonstrate the possibility to study pathway activation and interference in an iPSC model, that mimics the disease both with respect to the oncogenic signature and cell lineage commitment.