Circulating cell-free DNA (cfDNA) in patients with medullary thyroid carcinoma is characterized by specific fragmentation and methylation changes with diagnostic value.

Medullary Thyroid Carcinoma (MTC) is a rare neuroendocrine tumour whose diagnosis includes evaluating calcitonin serum levels, which can present fluctuations unrelated to MTC. Here, we investigated circulating DNA fragmentation and methylation changes as potential biomarkers using ddPCR on cell-free DNA (cfDNA) isolated from the plasma of MTC patients. For cfDNA fragmentation analysis, we investigated the fragment size distribution of a gene family and calculated short fragment fraction (SFF). Methylation analyses evaluated the methylation levels of CG_16698623, a CG dinucleotide in the MGMT gene that we found hypermethylated in MTC tissues by analyzing public databases. The SFF ratio and methylation of CG_16698623 were significantly increased in plasma from MTC patients at diagnosis, and patients with clinical remission or stable disease at follow-up showed no significant SFF difference compared with healthy subjects. Our data support the diagnostic value of cfDNA traits that could enable better management of MTC patients.

Molecular mechanisms of the tyrosine kinase inhibitor pralsetinib activity in in-vitro models of medullary thyroid carcinoma: Aberrant activation of the HH-Gli signaling pathway in acquired resistance.

Medullary thyroid carcinoma (MTC) is a malignant tumor with challenging management. Multi-targeted kinase inhibitors (MKI) and tyrosine-kinase inhibitors (TKI) with high specificity for RET protein are approved for advanced MTC treatment. However, their efficacy is hindered by evasion mechanisms of tumor cells. Thus, the aim of this study was the identification of an escape mechanism in MTC cells exposed to a highly selective RET TKI. TT cells were treated with TKI, MKI, and/or the HH-Gli inhibitors, GANT61 and Arsenic Trioxide (ATO), in the presence or absence of hypoxia. RET modifications, oncogenic signaling activation, proliferation and apoptosis were assessed. Additionally, cell modifications and HH-Gli activation were also evaluated in pralsetinib-resistant TT cells. Pralsetinib inhibited RET autophosphorylation and RET downstream pathways activation in normoxic and hypoxic conditions. Additionally, pralsetinib impaired proliferation, induced the activation of apoptosis and, in hypoxic cells, downregulated HIF-1α. Focusing on escape molecular mechanisms associated with therapy, we observed increased Gli1 levels in a subset of cells. Indeed, pralsetinib stimulated the re-localization of Gli1 into the cell nuclei. Treatment of TT cells with both pralsetinib and ATO resulted in Gli1 down-regulation and impaired cell viability. Moreover, pralsetinib-resistant cells confirmed Gli1 activation and up-regulation of its transcriptionally regulated target genes. Altogether, we showed that pralsetinib impairs MTC cell growth and induces cell death, also in hypoxic conditions. The HH-Gli pathway is a new molecular mechanism of escape to pralsetinib therapy that can be overcome through combined therapy.

Hedgehog-GLI and Notch Pathways Sustain Chemoresistance and Invasiveness in Colorectal Cancer and Their Inhibition Restores Chemotherapy Efficacy.

Colorectal cancer (CRC) is a leading cause of cancer-related mortality and chemoresistance is a major medical issue. The epithelial-to-mesenchymal transition (EMT) is the primary step in the emergence of the invasive phenotype and the Hedgehog-GLI (HH-GLI) and NOTCH signaling pathways are associated with poor prognosis and EMT in CRC. CRC cell lines harboring KRAS or BRAF mutations, grown as monolayers and organoids, were treated with the chemotherapeutic agent 5-Fluorouracil (5-FU) alone or combined with HH-GLI and NOTCH pathway inhibitors GANT61 and DAPT, or arsenic trioxide (ATO) to inhibit both pathways. Treatment with 5-FU led to the activation of HH-GLI and NOTCH pathways in both models. In KRAS mutant CRC, HH-GLI and NOTCH signaling activation co-operate to enhance chemoresistance and cell motility, while in BRAF mutant CRC, the HH-GLI pathway drives the chemoresistant and motile phenotype. We then showed that 5-FU promotes the mesenchymal and thus invasive phenotype in KRAS and BRAF mutant organoids and that chemosensitivity could be restored by targeting the HH-GLI pathway in BRAF mutant CRC or both HH-GLI and NOTCH pathways in KRAS mutant CRC. We suggest that in KRAS-driven CRC, the FDA-approved ATO acts as a chemotherapeutic sensitizer, whereas GANT61 is a promising chemotherapeutic sensitizer in BRAF-driven CRC.