Investigating the therapeutic potential of ATR, CHK1 and WEE1 inhibitors in cervical cancer

Abstract

Introduction: Cervical cancer is the 4th most common cause of cancer-related death in women. It is caused by infection with high-risk HPV (HR-HPV). Current therapy with cisplatin and radiotherapy acts by damaging DNA. The DNA damage response (DDR), critical for survival following endogenous and therapeutic DNA damage, comprises signalling to cell cycle checkpoints and DNA repair. HR-HPV inactivates p53 and pRB and thereby the GI/S checkpoint, making cervical cancer an ideal target for inhibition of intra-S and G2/M cell cycle checkpoints. This thesis directly compares the efficacy of inhibitors of the S and G2/M checkpoints: ATR, CHK1 and WEE1 as single agents and as sensitisers to cisplatin and ionising radiation in cervical cancer cell lines Methods: A panel of 6 cervical cancer cell lines with different histopathology and HPV status were used. DDR protein expression and inhibition of ATR, CHK1 and WEE1 by VE-821, PF477736 and MK-1775, respectively were measured by Western blot. Checkpoint proteins were measured in a TMA of human cervical cancer by IHC. Cytotoxicity of inhibitors and combinations with cisplatin or IR was determined by clonogenic assay. Cell cycle analysis with propidium iodide was used to investigate cell cycle changes. Results: The expression of DDR and checkpoint proteins varied in both cell lines and the TMA. There was a modest spectrum of sensitivity to cisplatin, IR and the inhibitors but the rank order was different for each agent, which was not related to the levels of DDR proteins in general but low ATM was associated with VE-821 sensitivity. The inhibitors were used at fixed concentrations for chemo- and radio-sensitisation studies: 1 µM VE821, 50 nM PF477736 and 100 nM MK-1775 reflecting their relative target inhibition potency and intrinsic cytotoxicity. Greater sensitisation was observed with cisplatin than IR, with VE-821 having the greatest and MK-1775 the least effect. Cisplatin caused S-phase accumulation that was reduced by the kinase inhibitors in 4/6 cell lines but increased in the other 2. The effects were more marked for VE-821 and PF-477736 vs MK-1775 and were not related to cisplatin sensitisation. Conclusions: Cytotoxicity and sensitisation effects were not explained by protein expressions or enzyme inhibition. The effect of the inhibitors on cisplatin-induced S-phase arrest varied across the cell line panel and did not correlate with sensitisation data. Analysis was hampered by the size of the panel and their similarity. Further work with a larger, more diverse panel of cell lines is required before the mechanisms and potential biomarkers of response to ATR, CHK1 and WEE1 inhibition in cervical cancer can be identified.