Targeting the DNA damage response kinase CHK1 in TP53-mutated thyroid cancer: in vitro studies

Objectives: Differentiated TCs are generally sensitive to first line treatments and tyrosine kinase inhibitors (TKIs). However, part of them along with undifferentiated TC, namely Anaplastic (ATC) and Poorly Differentiated (PDTC), are aggressive and show refractoriness to tyrosine-kinase inhibitors (TKIs) treatments. A correlation between resistance to TKIs and inactivating TP53 mutations was proven in TC by our group, consistent with data obtained in other tumors. To find a novel therapeutic strategy for aggressive TC, we aimed to investigate the DNA damage response (DDR) pathway, where p53 plays a crucial role. Indeed, since the combined lack of function of two DDR players can induce cancer cell death by synthetic lethality, the pharmacological inhibition of a DDR molecule in tumors lacking p53 function is a promising strategy currently studied and clinically tested in several solid and hematological malignancies. Methods: MMT cell viability assay, western blot and immunofluorescence analyses were used to characterize the DDR pathway in response to a DNA damaging agent in a panel of TC cell lines with known TP53 mutational status, and to evaluate the inhibitory effect of Prexasertib, a highly selective Chk1 kinase ATP-competitive inhibitor, on Chk1 kinase activity. Results: The in vitro characterization of the DDR showed the presence of genomic instability predominantly in TP53-mutated cell lines. Interestingly, Chk1 kinase resulted strongly activated in response to the genotoxic agent Doxorubicin (DOXO) in TP53-mutated cell lines (FRO, SW1736, B-CPAP and HTC/C3) as compared to the wild-type lines (TPC-1, K1 and IHH-4). Therefore, we tested the effect of Prexasertib (PX) on our TC cell lines and found that it was able to inhibit 50% of cell proliferation (IC50) at less than 10 nM concentration in all p53-deficient cell lines. Moreover, combined treatments with low concentration of PX (4nM) and IC25 DOXO showed remarkable decrease in TP53-mutated TC cell proliferation with respect to single treatments, without affecting healthy thyroid cell viability, as demonstrated treating patient-derived normal cultured thyrocytes. Conclusions: Our data showed, for the first time in TC, that Chk1 may be a suitable target for novel treatment strategies in p53-deficient aggressive TC.