Identification And Characterization Of Novel Targets In Papillary Thyroid Cancer (PTC)

Introduction: Papillary thyroid cancer (PTC) is the most common type of endocrine malignancy and accounts for 96% of total new endocrine cancers and 68.8% deaths due to endocrine cancers. Mutations in BRAF (mostly V600E), RAS or gene fusions involving RET and NTRK1 are the most common genetic alterations identified in PTC. Though surgical resection followed by radioactive-iodine therapy (RAI) usually results in good prognosis of PTC, factors like increase in disease recurrence, distant metastasis and resistance to RAI in advanced disease conditions have been associated with patient morbidity and mortality which emphasises the need for more effective treatment methods. Identifying and understanding the molecular pathogenesis underlying thyroid cancer is important for the development of precision medicine and personalized therapeutics. The main aim of the study is the identification and validation of novel genetic alterations in PTC by employing multidimensional omics approaches and to develop rational inhibitors.

Materials and methods: From a set of patients whose PTC did not harbour any known BRAF or RAS mutations, a 35 years old male patient’s normal, primary tumor and metastatic tissues were selected for both genomic and label free quantitative proteomic analysis. We identified a novel RET gene fusion in this patient and the oncogenic ability of this novel gene fusion was tested in transformation assays. We employed multiple biochemical and cell biological assays to characterize the signalling pathways activated by this novel gene fusion in human thyroid cells. Finally, we employed small molecule inhibitors against selected targets and tested their effects in colony formation assays.

Results and discussion: Stable expression of the novel RET fusion gene activated several oncogenic signalling pathways and transformed immortalized human thyroid cells in a kinase dependent manner. The novel RET fusion exhibited high kinase activity and formed dimers and oligomers partially in a PB1 domain dependent manner. Quantitative proteomics studies revealed the upregulation of proteins involved in the ubiquitination machinery including HECT carrying E3 ligase HUWE1 and DUBs like Usp9X and UBP7 in the tumour and metastatic lesions. Inhibition of RET, HUWE and DUBs by small molecule inhibitors significantly reduced RET-mediated oncogenesis.

Conclusion: Apart from unveiling a novel oncogenic RET fusion in PTCs, our data open a novel avenue of targeting ubiquitin signaling machinery in human PTCs.