TARGETING THE DNA DAMAGE RESPONSE PATHWAY FOR THE TREATMENT OF AGGRESSIVE THYROID CANCERS, IN VITRO STUDIES

Background. Differentiated thyroid cancers (TC) are generally sensitive to first line treatments and tyrosine kinase inhibitors (TKIs). Though, part of them as well as Poorly Differentiated and Anaplastic TC are particularly aggressive and refractory both to first-line and TKI treatments, such as Lenvatinib (LENV). In other aggressive tumours (NSCLC, HCC, CML), TKI resistance is known to be correlated with the presence of TP53 gene inactivating mutations which also are frequently harboured by aggressive TC. Research issue. Aiming to find a novel treatment for aggressive TC, we investigated the DNA damage response (DDR) pathway where p53 plays a crucial role. Indeed, the pharmacological inhibition of DDR kinases for the treatment of p53-defective tumours is a promising strategy, known as synthetic lethality, currently studied and clinically tested in several cancers either in single or combined treatments with chemotherapy or targeted therapies. Methodology. MTT-based cell viability assay, Western Blot and immunofluorescence analyses were performed to characterize the DDR pathway in untreated condition and in response to Doxorubicin (DX), an agent that induces DNA double-strand breaks, in a panel of TC cell lines with known TP53 alterations. These methods were also used to evaluate the effect of a selective Chk1 kinase inhibitor, Prexasertib (PX), currently employed in ongoing phase II clinical trials for other cancers treatment, which was used to treat cell lines either alone or in combination with DX or LENV. Flowcytometry analysis was used to evaluate cell cycle variations upon single and combined treatments. Results. A significant association between resistance to TKI treatments and the presence of TP53 inactivating mutations was found in a cohort of 30 patients with aggressive TC. This data was further corroborated by in vitro analysis with LENV treatment, showing that also TP53-mutated TC cell lines (SW1736, B-CPAP, HTC/C3) proved resistant to LENV. Instead, among the TP53-wildtype cells, only TPC-1 cell line proved fully LENV sensitive, while IHH- 4, characterized by alterations in p300/CBP complex regulating p53 function, showed low sensitivity to LENV, and FRO, lacking p53 expression, displayed resistance to LENV. Therefore, we sought to further investigate in vitro the DNA damage response (DDR) pathway, where p53 plays a central role, aiming to find a novel molecular target for TKI- resistant TC treatment. The initial characterization of p53-defective and LENV-resistant TC cells (FRO, SW1736, B-CPAP, HTC/C3) showed a higher degree of genomic instability and variable levels of the DDR kinase proteins ATR, Chk1 and ATM, Chk2 as opposed to the p53-proficient and LENV- sensitive cell lines (TPC-1, IHH-4). Notably, DX treatment, generally inducing ATM/Chk2 kinases activation, caused in p53- defective TC cells also the peculiar activation of ATR/Chk1 kinases as an alternative way to induce cell cycle arrest for the induction of DNA damage repair in the absence of p53 function. Therefore, Chk1 kinase inhibition was considered a valid molecular target treatment for these p53-defective TC cells, and Prexasertib (PX) inhibitor was selected, already proving efficacious in several tumours in phase I and II clinical trials. In accordance with the synthetic lethality principle, p53-defective TC cells proved more sensitive to PX treatment than the p53-proficient ones. Overall, in p53-defective TC cells less than 10 nM PX was able to induce an IC50 antiproliferative effect associated with DNA damage accumulation, except for B-CPAP cells showing resistance to PX treatment. Up to 10,6 nM PX was able to induce an increase of p53-defective cells in S phase at different time points, consequently to the accumulation of unresolved DNA damages, and resulted in apoptosis as assayed by WB of Caspase3 and PARP cleavages. Moreover, when inducing DNA damages with DX together with Chk1 inhibition by PX treatment, yet the lowest doses (4 nM PX and cell-specific IC25 DX) were sufficient to induce in p53-defective TC cells a more extensive reduction in cell viability and specular increase of apoptosis with respect to single treatments, remarkably including the PX-resistant cell line B-CPAP. As expected, PX and DX combined treatments did not affect healthy thyrocytes viability. Interestingly, LENV effects on cell viability resulted to be potentiated by 10,6 nM PX only in a p53-proficient and LENV-sensitive TC cell line (IHH-4), showing an increased number of cells in S phase and a similar tendency in preG1 phase, suggesting cell death. The p53 and Chk1 functionally related gene UHRF1, regulating under p53 control cell cycle progression and VEGF expression, resulted to be overexpressed in samples derived from TKI resistant patients, thus providing mechanistic and prognostic insights in TC patients response to TKIs. Conclusions This study uncovers the potential of Prexasertib, a Chk1-inhibitor, as a novel treatment option for p53-defective aggressive TC that are resistant to TKIs. Furthermore, it sheds light on the possible prognostic value of p53 and UHRF1 as predictive biomarkers of TKI response.